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Sample records for mylitta cytoplasmic polyhedrosis

  1. Genome segment 5 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes a bona fide guanylyltransferase

    PubMed Central

    2014-01-01

    Background Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects non mulberry Indian silk worm, Antheraea mylitta, and contains eleven segmented double stranded RNA in its genome (S1-S11). Some of its genome segments (S1-S3, and S6-S11) have been previously characterized but genome segment encoding the viral guanylyltransferase which helps in RNA capping has not been characterized. Results In this study genome segment 5 (S5) of AmCPV was converted to cDNA, cloned and sequenced. S5 consisted of 2180 nucleotides, with one long ORF of 1818 nucleotides and could encode a protein of 606 amino acids with molecular mass of ~65 kDa (p65). Bioinformatics analysis showed presence of KLRS and HxnH motifs as observed in some other reoviral guanylyltransferase and suggests that S5 may encodes viral guanylyltransferase. The ORF of S5 was expressed in E. coli as 65 kDa his tagged fusion protein, purified through Ni-NTA chromatography and polyclonal antibody was raised. Immunoblot analysis of virion particles with the purified antibody showed specific immunoreactive band and suggests p65 as a viral structural protein. Functional analysis showed that recombinant p65 possesses guanylyltransferase activity, and transfers GMP moiety to the 5' diphosphate (A/G) ended viral RNA after the formation of p65-GMP complex for capping. Kinetic analysis showed Km of this enzyme for GTP and RNA was 34.24 uM and 98.35 nM, respectively. Site directed mutagenesis at K21A in KLRS motif, and H93A or H105A in HxnH motif completely abolished the autoguanylylation activity and indicates importance of these residues at these sites. Thermodynamic analysis showed p65-GTP interaction was primarily driven by enthalpy (ΔH = -399.1 ± 4.1 kJ/mol) whereas the p65-RNA interaction by favorable entropy (0.043 ± 0.0049 kJ/ mol). Conclusion Viral capping enzymes play a critical role in the post transcriptional or post replication modification in case of

  2. Genome segment 4 of Antheraea mylitta cytoplasmic polyhedrosis virus encodes RNA triphosphatase and methyltransferases.

    PubMed

    Biswas, Poulomi; Kundu, Anirban; Ghosh, Ananta Kumar

    2015-01-01

    Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) genome segment S4 showed that it consists of 3410 nt with a single ORF of 1110 aa which could encode a protein of ~127 kDa (p127). Bioinformatics analysis showed the presence of a 5' RNA triphosphatase (RTPase) domain (LRDR), a S-adenosyl-l-methionine (SAM)-binding (GxGxG) motif and the KDKE tetrad of 2'-O-methyltransferase (MTase), which suggested that S4 may encode RTPase and MTase. The ORF of S4 was expressed in Escherichia coli as a His-tagged fusion protein and purified by nickel-nitrilotriacetic acid affinity chromatography. Biochemical analysis of recombinant p127 showed its RTPase as well as SAM-dependent guanine N(7)-and ribose 2'-O-MTase activities. A MTase assay using in vitro transcribed AmCPV S2 RNA having a 5' G*pppG end showed that guanine N(7) methylation occurred prior to the ribose 2'-O methylation to yield a m(7)GpppG/m(7)GpppGm RNA cap. Mutagenesis of the SAM-binding (GxGxG) motif (G831A) completely abolished N(7)- and 2'-O-MTase activities, indicating the importance of these residues for capping. From the kinetic analysis, the Km values of N(7)-MTase for SAM and RNA were calculated as 4.41 and 0.39 µM, respectively. These results suggested that AmCPV S4-encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5' end of viral RNA. PMID:25228490

  3. Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

    SciTech Connect

    Ghorai, Suvankar; Chakrabarti, Mrinmay; Roy, Sobhan; Chavali, Venkata Ramana Murthy; Bagchi, Abhisek; Ghosh, Ananta Kumar

    2010-08-15

    Genome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123 kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123 kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37 deg. C at pH 6.0 in the presence of 3 mM MgCl{sub 2.} Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents.

  4. Transcriptome analysis of interactions between silkworm and cytoplasmic polyhedrosis virus

    PubMed Central

    Jiang, Liang; Peng, Zhengwen; Guo, Youbing; Cheng, Tingcai; Guo, Huizhen; Sun, Qiang; Huang, Chunlin; Zhao, Ping; Xia, Qingyou

    2016-01-01

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects silkworm midgut (MG) and multiplication occurs mainly in posterior midgut (PM). In this study, MG and fat body (FB) were extracted at 0, 3, 24, and 72 h after BmCPV infection. The total sequence reads of each sample were more than 1510000, and the mapping ratio exceeded 95.3%. Upregulated transcripts increased in MG during the infection process. Gene ontology (GO) categories showed that antioxidants were all upregulated in FB but not in MG. BGI001299, BGI014434, BGI012068, and BGI009201 were MG-specific genes with transmembrane transport function, the expression of which were induced by BmCPV. BGI001299, BGI014434, and BGI012068 expressed in entire MG and may be involved in BmCPV invasion. BGI009201 expressed only in PM and may be necessary for BmCPV proliferation. BmPGRP-S2 and BGI012452 (a putative serine protease) were induced by BmCPV and may be involved in immune defense against BmCPV. The expression level of BmCPV S1, S2, S3, S6, and S7 was high and there was no expression of S9 in MG 72 h, implying that the expression time of structural protein coding genes is earlier. These results provide insights into the mechanism of BmCPV infection and host defense. PMID:27118345

  5. Characterization of a Ribonucleic Acid Polymerase Activity Associated with Purified Cytoplasmic Polyhedrosis Virus of the Silkworm Bombyx mori1

    PubMed Central

    Lewandowski, L. J.; Kalmakoff, J.; Tanada, Y.

    1969-01-01

    Purified cytoplasmic-polyhedrosis virus has been found to have associated with it a polymerase activity capable of catalyzing the synthesis of virus-specific, single-stranded ribonucleic acid (RNA) from the double-stranded RNA genome. PMID:16789118

  6. A cytoplasmic polyhedrosis virus isolated from the pine processionary caterpillar, Thaumetopoea pityocampa.

    PubMed

    Ince, Ikbal Agah; Demir, Ismail; Demirbag, Zihni; Nalcacioglu, Remziye

    2007-04-01

    A cytoplasmic polyhedrosis virus (CPV) was isolated from the larvae of Thaumetopoea pityocampa and shown to cause an infection of midgut cells. This viral infection revealed several important diagnostic symptoms, including discoloration of the posterior midgut, reduced feeding, and extended development time of the larvae. The virus infection is lethal to Thaumetopoea pityocampa, and with the increasing doses kills the larvae within 4-5 days post infection. Electron microscopy studies showed typical cytoplasmic polyhedral inclusion bodies that are icosahedral, and ranged from 2.4 to 5.3 microm in diameter. Electrophoretic analysis of the RNA genome showed that the virus has a genome composed of 10 equimolar RNA segments with the sizes of 3,907, 3,716, 3,628, 3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp, respectively. Based on morphology and nucleic acid analysis, this virus was named Thaumetopoea pityocampa cytoplasmic polyhedrosis virus (TpCPV), and belongs to the genus Cypovirus, family Reoviridae. PMID:18051275

  7. X-ray powder pattern analysis of cytoplasmic polyhedrosis virus inclusion bodies

    SciTech Connect

    Di, X.; Sun, Y.K.; McCrae, M.A.; Rossmann, M.G. )

    1991-01-01

    Cytoplasmic polyhedrosis virus is an insect reovirus which is occluded in crystalline inclusion bodies that form in the mid-gut of certain insects. Inclusion bodies of cytoplasmic polyhedrosis virus from Bombyx mori were isolated and purified. These crystalline bodies, about 1-3 microns in linear size, were compacted in a capillary tube while immersed in buffer. X-ray diffraction photographs showed powder rings, extending to 8.2 A resolution, which could be indexed with a cell measuring a = b = 49.9 +/- 0.4 A, c = 41.5 +/- 0.4 A, alpha = beta = gamma = 90 degrees. The polyhedrin protein, which is the major component of the inclusion body, has a molecular weight of about 30,000 daltons and, hence, there are probably two molecules in the unit cell. Thus, the unit cell is monoclinic or possibly triclinic. A Patterson derived from the measured powder pattern intensities, assuming monoclinic symmetry, could be interpreted in terms of a molecule with two larger globes. Such a structure is roughly consistent with the breakdown of the polyhedrin into two larger fragments of molecular weight 17,000 and 14,000 when raising the pH to near 10. Under these conditions the inclusion bodies disintegrate, releasing virus and catalyzing the proteolysis of the polyhedrin.

  8. Transcriptome Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

    PubMed Central

    Kolliopoulou, Anna; Van Nieuwerburgh, Filip; Stravopodis, Dimitrios J.; Deforce, Dieter; Swevers, Luc; Smagghe, Guy

    2015-01-01

    Many insects can be persistently infected with viruses but do not show any obvious adverse effects with respect to physiology, development or reproduction. Here, Bombyx mori strain Daizo, persistently infected with cytoplasmic polyhedrosis virus (BmCPV), was used to study the host’s transcriptional response after pathogenic infection with the same virus in midgut tissue of larvae persistently and pathogenically infected as 2nd and 4th instars. Next generation sequencing revealed that from 13,769 expressed genes, 167 were upregulated and 141 downregulated in both larval instars following pathogenic infection. Several genes that could possibly be involved in B. mori immune response against BmCPV or that may be induced by the virus in order to increase infectivity were identified, whereas classification of differentially expressed transcripts (confirmed by qRT-PCR) resulted in gene categories related to physical barriers, immune responses, proteolytic / metabolic enzymes, heat-shock proteins, hormonal signaling and uncharacterized proteins. Comparison of our data with the available literature (pathogenic infection of persistently vs. non-persistently infected larvae) unveiled various similarities of response in both cases, which suggests that pre-existing persistent infection does not affect in a major way the transcriptome response against pathogenic infection. To investigate the possible host’s RNAi response against BmCPV challenge, the differential expression of RNAi-related genes and the accumulation of viral small RNAs (vsRNAs) were studied. During pathogenic infection, siRNA-like traces like the 2-fold up-regulation of the core RNAi genes Ago-2 and Dcr-2 as well as a peak of 20 nt small RNAs were observed. Interestingly, vsRNAs of the same size were detected at lower rates in persistently infected larvae. Collectively, our data provide an initial assessment of the relative significance of persistent infection of silkworm larvae on the host response following

  9. Roles of miR-278-3p in IBP2 regulation and Bombyx mori cytoplasmic polyhedrosis virus replication.

    PubMed

    Wu, Ping; Qin, Guangxing; Qian, Heying; Chen, Tao; Guo, Xijie

    2016-01-10

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major viral pathogen of silkworm and remains a big challenge to the sericultural industry. Insulin-related peptide binding protein 2 (IBP2) gene, induced by BmCPV infection may play an important role in B. mori immune response. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the post-transcriptional level and play an important role in various processes, including immunity and antiviral response. In this study, we identified IBP2 as one of the targets for miR-278-3p by using luciferase reporter assay. Overexpression of miR-278-3p negatively regulates the expression of IBP2 in silkworm larvae and positively regulates the mRNA transcript level of BmCPV. Our results suggest that miR-278-3p may play an important role in BmCPV replication. It's the first report on bmo-miRNAs in response to BmCPV infection and could provide a new clue to explore the molecular mechanism of BmCPV infection and host immunity. PMID:26348138

  10. dsRNA interference on expression of a RNA-dependent RNA polymerase gene of Bombyx mori cytoplasmic polyhedrosis virus.

    PubMed

    Pan, Zhong-Hua; Gao, Kun; Hou, Cheng-Xiang; Wu, Ping; Qin, Guang-Xing; Geng, Tao; Guo, Xi-Jie

    2015-07-01

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the major viral pathogens in silkworm. Its infection often results in significant losses to sericulture. Studies have demonstrated that RNAi is one of the important anti-viral mechanisms in organisms. In this study, three dsRNAs targeting the RNA-dependent RNA polymerase (RDRP) gene of BmCPV were designed and synthesized with 2'-F modification to explore their interference effects on BmCPV replication in silkworm larvae. The results showed that injecting dsRNA in the dosage of 4-6 ng per mg body weight into the 5th instar larvae can interfere with the BmCPV-RDRP expression by 93% after virus infection and by 99.9% before virus infection. In addition, the expression of two viral structural protein genes (genome RNA segments 1 and 5) was also decreased with the decrease of RDRP expression, suggesting that RNAi interference of BmCPV-RDRP expression could affect viral replication. The study provides an effective method for investigating virus replication as well as the virus-host interactions in the silkworm larvae using dsRNA. PMID:25839934

  11. Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

    PubMed Central

    Van Nieuwerburgh, Filip; Kolliopoulou, Anna; Apostolou-Karampelis, Konstantinos; Head, Steven R.; Deforce, Dieter; Smagghe, Guy; Swevers, Luc

    2015-01-01

    ABSTRACT The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera. IMPORTANCE This work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented double-stranded RNA (dsRNA) virus (CPV; Reoviridae) and highlights the importance of viral small-RNA (vsRNA) analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20-nucleotide vsRNAs or based on the activity of an uncharacterized endo-RNase that cleaves the viral RNA substrate at a degenerate motif. The analysis also indicates the existence of a degradation pathway that targets the positive strand of segment 10. PMID

  12. Reconstitution of the RNA-dependent RNA polymerase activity of Antheraea mylitta cypovirus in vitro using separately expressed different functional domains of the enzyme.

    PubMed

    Kundu, Anirban; Roychowdhury, Amlan; Bose, Madhuparna; Das, Amit Kumar; Ghosh, Ananta K

    2016-07-01

    Antheraea mylitta cytoplasmic polyhedrosis virus is a segmented dsRNA virus of the family Reoviridae. Segment 2 (S2)-encoded RNA-dependent RNA polymerase (RdRp) helps the virus to propagate its genome in the host cell of the silkworm, Antheraea mylitta. Cloning, expression, purification and functional analysis of individual domains of RdRp have demonstrated that the purified domains interact in vitro. The central polymerase domain (PD) shows nucleotide binding properties, but neither the N-terminal domain (NTD) nor the C-terminal domain (CTD). Isolated PD does not exhibit RdRp activity but this activity can be reconstituted when all three domains are included in the reaction mixture. Molecular dynamics simulation suggests that the isolated PD has increased internal motions in comparison to when it is associated with the NTD and CTD. The motions of the separated PD may lead to the formation of a less accessible RNA template-binding channel and, thus, impair RdRp activity. PMID:27008451

  13. Characterization of Nuclear Polyhedrosis Virus DNAs

    PubMed Central

    Summers, Max D.; Anderson, David L.

    1973-01-01

    The nuclear polyhedrosis virus DNAs characterized and compared in this study consist of the singly-enveloped nucleocapsids (SNPV) of Trichoplusia ni and the bundles of nucleocapsids common to a single envelope (MNPV) from Spodoptera frugiperda and Rachiplusia ou. The SNPV and MNPV DNAs are very similar in hydrodynamic properties and molecular weights. In addition, the NPV DNAs are similar in size to those extracted from the granulosis viruses that infect T. ni and S. frugiperda. As isolated from purified virus or directly from occluded virus, the nuclear polyhedrosis virus DNAs consist of a mixture of about 20 to 30% double-stranded covalently closed molecules and approximately 60% relaxed circles, with less than 10% in linear duplex form. The molecular weights of all nuclear polyhedrosis virus DNAs as compared in this study are slightly smaller than those of T4 bacteriophage DNA and perhaps slightly smaller than those of the granulosis virus DNAs. The best estimates of these molecular weights by neutral sucrose sedimentation for the nuclear polyhedrosis viruses range from 90 to 100 × 106 relative to a size of 108 × 106 for T4 DNA. The base compositions of the nuclear polyhedrosis viruses that infect T. ni and S. frugiperda are compared with the respective insect host DNAs. Images PMID:4761726

  14. Structure of Cytoplasmic Polyhedrosis Virus from Bombyx mori.

    PubMed

    Yu, Xue-Kui; Lu, Xin-Yin; Zhang, Hong; Zhou, Zhen-Hong; Zhang, Qin-Fen; Zhang, Xing; Zhang, Jing-Qiang

    1999-01-01

    The structures of full and empty capsids of CPV were studied by negative staining and electron cryomicroscopy and computer reconstruction techniques. By comparing the structures and biochemical compositions, the CPV was identified as a single-layered capsid with its five structural proteins located on it. This single capsid is arranged according to T=1 icosahedral symmetry with 12 turret-like spikes at its icosahedral vertices. The empty and full CPV show identical capsid but differ inside. The dense and ordered genomic dsRNA is located inside the full CPV. The internal space of the empty CPV has almost no electron density except for 12 electron densities attributed the transcriptional enzyme complexes extending inward from the base part of CPV spikes. PMID:12114971

  15. Genetic analysis of Indian tasar silkmoth (Antheraea mylitta) populations.

    PubMed

    Chakraborty, Saikat; Muthulakshmi, M; Vardhini, Deena; Jayaprakash, P; Nagaraju, J; Arunkumar, K P

    2015-01-01

    Indian tasar silkmoth, Antheraea mylitta is an economically important wild silkmoth species distributed across India. A number of morphologically and ethologically well-defined ecotypes are known for this species that differ in their primary food plant specificity. Most of these ecotypes do not interbreed in nature, but are able to produce offspring under captive conditions. Microsatellite markers were developed for A. mylitta, and out of these, ten well-behaved microsatellite loci were used to analyze the population structure of different ecoraces. A total of 154 individual moths belonging to eight different ecoraces, were screened at each locus. Hierarchical analysis of population structure using Analysis of MOlecular VAriance (AMOVA) revealed significant structuring (FST = 0.154) and considerable inbreeding (FIS = 0.505). A significant isolation by distance was also observed. The number of possible population clusters was investigated using distance method, Bayesian algorithm and self organization maps (SOM). The first two methods revealed two distinct clusters, whereas the SOM showed the different ecoraces not to be clearly differentiated. These results suggest that although there is a large degree of phenotypic variation among the different ecoraces of A. mylitta, genetically they are not very different, and the phenotypic differences may largely be a result of their respective ecology. PMID:26510465

  16. Genetic analysis of Indian tasar silkmoth (Antheraea mylitta) populations

    PubMed Central

    Chakraborty, Saikat; Muthulakshmi, M; Vardhini, Deena; Jayaprakash, P; Nagaraju, J; Arunkumar, K. P.

    2015-01-01

    Indian tasar silkmoth, Antheraea mylitta is an economically important wild silkmoth species distributed across India. A number of morphologically and ethologically well-defined ecotypes are known for this species that differ in their primary food plant specificity. Most of these ecotypes do not interbreed in nature, but are able to produce offspring under captive conditions. Microsatellite markers were developed for A. mylitta, and out of these, ten well-behaved microsatellite loci were used to analyze the population structure of different ecoraces. A total of 154 individual moths belonging to eight different ecoraces, were screened at each locus. Hierarchical analysis of population structure using Analysis of MOlecular VAriance (AMOVA) revealed significant structuring (FST = 0.154) and considerable inbreeding (FIS = 0.505). A significant isolation by distance was also observed. The number of possible population clusters was investigated using distance method, Bayesian algorithm and self organization maps (SOM). The first two methods revealed two distinct clusters, whereas the SOM showed the different ecoraces not to be clearly differentiated. These results suggest that although there is a large degree of phenotypic variation among the different ecoraces of A. mylitta, genetically they are not very different, and the phenotypic differences may largely be a result of their respective ecology. PMID:26510465

  17. 40 CFR 180.1027 - Nuclear polyhedrosis virus of Heliothis zea; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Nuclear polyhedrosis virus of... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1027 Nuclear polyhedrosis virus of... Heliothis zea nuclear polyhedrosis virus (HzSNPV). The identity of the seed virus must be assured...

  18. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all...

  19. 40 CFR 180.1027 - Nuclear polyhedrosis virus of Heliothis zea; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Nuclear polyhedrosis virus of... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1027 Nuclear polyhedrosis virus of... Heliothis zea nuclear polyhedrosis virus (HzSNPV). The identity of the seed virus must be assured...

  20. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all...

  1. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all...

  2. 40 CFR 180.1027 - Nuclear polyhedrosis virus of Heliothis zea; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Nuclear polyhedrosis virus of... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1027 Nuclear polyhedrosis virus of... Heliothis zea nuclear polyhedrosis virus (HzSNPV). The identity of the seed virus must be assured...

  3. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all...

  4. 40 CFR 180.1149 - Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... polyhedrosis virus of Anagrapha falcifera; exemption from the requirement of a tolerance. 180.1149 Section 180... Inclusion bodies of the multi-nuclear polyhedrosis virus of Anagrapha falcifera; exemption from the... polyhedrosis virus of Anagrapha falcifera is exempted from the requirement of a tolerance in or on all...

  5. 40 CFR 180.1027 - Nuclear polyhedrosis virus of Heliothis zea; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Nuclear polyhedrosis virus of... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1027 Nuclear polyhedrosis virus of... Heliothis zea nuclear polyhedrosis virus (HzSNPV). The identity of the seed virus must be assured...

  6. 40 CFR 180.1027 - Nuclear polyhedrosis virus of Heliothis zea; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Nuclear polyhedrosis virus of... PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1027 Nuclear polyhedrosis virus of... Heliothis zea nuclear polyhedrosis virus (HzSNPV). The identity of the seed virus must be assured...

  7. Safety evaluation of nuclear polyhedrosis virus replication in pigs.

    PubMed Central

    Döller, G; Gröner, A; Straub, O C

    1983-01-01

    To evaluate the hygienic risk involved in using baculoviruses for insect pest control, safety studies are required. Pigs were chosen as representative test animals of commercial and agricultural importance. The tests were aimed at detecting virus propagation, immune reactions, and signs of acute infection (changes in body temperature and hematology profile, swelling of lymph nodes). Four of five animals inoculated with nuclear polyhedrosis virus showed a slight temperature rise at day 2 postinfection. After day 4 postinfection, no differences between infected animals and controls were observed. In the bioassay, virus activity could be recovered from fecal samples; however, no activity was found in organ extracts. The data did not indicate hygienic risks involved in the application of nuclear polyhedrosis virus, especially that from Mamestra brassicae, in biological pest control. PMID:6344789

  8. Safety evaluation of nuclear polyhedrosis virus replication in pigs.

    PubMed

    Döller, G; Gröner, A; Straub, O C

    1983-04-01

    To evaluate the hygienic risk involved in using baculoviruses for insect pest control, safety studies are required. Pigs were chosen as representative test animals of commercial and agricultural importance. The tests were aimed at detecting virus propagation, immune reactions, and signs of acute infection (changes in body temperature and hematology profile, swelling of lymph nodes). Four of five animals inoculated with nuclear polyhedrosis virus showed a slight temperature rise at day 2 postinfection. After day 4 postinfection, no differences between infected animals and controls were observed. In the bioassay, virus activity could be recovered from fecal samples; however, no activity was found in organ extracts. The data did not indicate hygienic risks involved in the application of nuclear polyhedrosis virus, especially that from Mamestra brassicae, in biological pest control. PMID:6344789

  9. Attacin gene sequence variations in different ecoraces of tasar silkworm Antheraea mylitta

    PubMed Central

    Sudha, Rati; Murthy, Geetha N; Awasthi, Arvind K; Ponnuvel, Kangayam M

    2015-01-01

    Attacin gene exists as paralogous conversion and is being used for identification of strain variations in insects based on the sequence variation. Hence, a study was undertaken to analyze the sequence variation of the attacin gene isoforms in the tasar silkworm Anthereae mylitta that exists in the form of different ecoraces depending upon the environment, food plant and location. Comparison of the previously reported attacin sequences with the DNA sequences of attacin A and B genes revealed six amino acid substitutions among the sequences of the ecoraces which however did not affect the functional domain of Attacin. The generated dendrogram clearly indicated unique branches for each ecorace with two separate gene clusters for attacin A and B. The Sarihan ecorace formed a separate sub-group under both the gene clusters. The present study also revealed the presence of Attacin_N Superfamily domain exclusively in Exon I separated from the Attacin_C Superfamily domain that was present in Exon II and part of Exon III, a prominent character of attacin gene. The phylogenetic reconstruction analysis of attacin gene in A.mylitta supported the common evolutionary origin of attacin genes belonging to the Lepidoteran and Dipteran families that formed two separate clusters. PMID:26664033

  10. Generation of cytotoxic molecules and oxidative stress in haemolymph of pebrinised tasar silkworm Antheraea mylitta Drury.

    PubMed

    Jena, Karmabeer; Pandey, Jay Prakash; Priya, Anshu; Kundu, Papri; Sinha, Ajit Kumar; Yadav, Harendra; Sahay, Alok

    2016-01-01

    The present study was carried out to investigate the effects of microsporidial infection on redox regulation mechanism and oxidative stress in tasar silkworm Antheraea mylitta. High level of superoxide radical (p < 0.05), nitric oxide (p < 0.001) and lipid peroxidation (p < 0.001) was observed in haemolymph of pebrinised larvae, which indicated the resultant generation of cytotoxic molecules and oxidative damage. Increased phenol oxidase (PO) activity in haemolymph of pebrinised larvae indicated the activation of immune defences during pathological conditions. In addition, higher level of glutathione-S-tranferase (GST) activity and reduced glutathione (GSH) level observed in pebrinised larvae indicated adaptive behaviour of tissue against toxic oxyradicals (p < 0.05). Conversely, low level of ascorbic acid (ASA) content suggested that the larvae might have used these compounds to counteract stress in tissues or low uptake under microspridial infection (p < 0.05). Present findings provide new insights into the cellular and biochemical bases of host-pathogen interactions in tasar silkworm A. mylitta. PMID:26930859

  11. Estimation of amino acids, urea and uric acid in tasar silkworm, Antheraea mylitta Drury.

    PubMed

    Shamitha, G; Rao, A Purushotham

    2008-11-01

    The tasar silkworm, Antheraea mylitta Drury, Andhra local ecorace is an exclusive race of Andhra Pradesh. It is on the verge of extinction due to difficulty of acclimatisation at breeding and rearing stages. As an attempt to protect this race, a method of total indoor rearing has been done. In this context, the estimation of free amino acids, excretory products- urea and uric acid were compared during the fourth and fifth instars of tasar silkworm, reared under outdoor and indoor conditions. The study has revealed that amino acids decreased in the fat body in outdoor and indoor reared larvae in contrast to that in the haemolymph where it has gradually increased from first to third crops. This is an important finding as it reveals that indoor worms seem to adopt proteolytic activity in the haemolymph. Secondly, in the fifth instar the excretory products are more compared to fourth instar in the indoor reared worms. During fifth instar, formation of nitrogenous products lessens as silk synthesis enhances. The present study reveals that decrease in uric acid in fifth instar implies increase in growth rate and silk synthesis in both outdoor and indoor worms. The findings of the present investigation is helpful in the conservation and protection of the A. mylitta, Andhra local ecorace. PMID:19297987

  12. ANALYSIS OF THE 'SPODOPTERA FRUGIPERDA' NUCLEAR POLYHEDROSIS VIRUS GENOME BY RESTRICTION ENDONUCLEASES AND ELECTRON MICROSCOPY

    EPA Science Inventory

    Restriction endonuclease analysis was used to differentiate between four strains of Spodoptera frugiperda nuclear polyhedrosis virus from different geographical areas. In addition, partial denaturation was performed, and a partial denaturation map was constructed for the Ohio str...

  13. [Comparative biochemical studies of polyhedral proteins of nuclear polyhedrosis viruses].

    PubMed

    Kozlov, E A; Levitina, T L; Gusak, N M; Larionov, G V; Veremeĭchenko, S N

    1978-12-01

    Using disc polyacrylamide gel electrophoresis, the molecular weights of polyhedral proteins of nuclear polyhedrosis viruses (NPV) of Porthetria dispar, Mamestra brassicae, and Aporia crataegi were found to be 28000 +/- 3000. It was shown that NPV polyhedra of Bombyx mori, Galleria mellonella, P. dispar, and M. brassicae contain a protease. During dissolution of the polyhedra at pH 10,5 this protease specifically cleaves the matrix protein into 2--5 fragments. The amino acid compositions of NPV polyhedral proteins of P. dispar, M. brassicae, A. crataegi, Hyphantria cunae were shown to be very similar. It was found that tyrosine is a C-terminal amino acid of NPV polyhedral proteins of P. dispar, M. brassicae, and A. crataegi. PMID:33725

  14. ISSR profiling of genetic variability in the ecotypes of Antheraea mylitta Drury, the tropical Tasar silkworm.

    PubMed

    Chatterjee, S N; Vijayan, K; Roy, G C; Nair, C V

    2004-02-01

    Antheraea mylitta, Drury, the semi-wild silk-producing lepidopteran insect commonly known as tasar silkworm is unique to India and is distributed over a wide tropical forest range covering the states of Andhra Pradesh, Bihar, Chhattisgarh, Madnya Pradesh, Maharashtra, Orissa and Uttaranchal. The populations found in different areas are know by their specific local names and are considered as different ecotypes, but it is difficult to separate the populations on the basis of morphological and life-cycle traits and thus molecular characterization was attempted. The present communication relates to the results obtained from the analysis of polymorphism unraveled by twelve ISSR primers for 11 populations of A. mylitta belonging to six ecotypes and 41 individuals of "Railey"--ecotype collected from five zones of Dandakarnya forest in Madnya Pradesh. This communication, further, presents molecular evidences on genetic differences between eleven ecotype populations and highlights the genotypic diversification of a single ecotype into further separate discrete gene pools. The canonical discriminant function analysis revealed grouping of the five populations of Railey ecotype into two "clumps", while accessions of other ecotypes stood separated from each other. Thr "Railey" populations on detailed study, further, revealed separation of two (Tokapal and Nangur) populations into discrete gene pools and the other three (Kondagaon, Darba and Tongpal) populations, in spite of larger geographic distance between them, overlapped one on the other. The analysis also identified nine markers, which can be utilized to characterize specific population and will be of help to follow the ongoing genetic changes triggered by various ecological factors and human influences on the "Railey" ecotype. PMID:15065428

  15. 40 CFR 180.1118 - Spodoptera exigua nuclear polyhedrosis virus; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... virus; exemption from the requirement of a tolerance. 180.1118 Section 180.1118 Protection of... polyhedrosis virus; exemption from the requirement of a tolerance. An exemption from the requirement of a tolerance is established for the microbial pest control agent Spodoptera exigua nuclear polyhedrosis...

  16. 40 CFR 180.1118 - Spodoptera exigua nuclear polyhedrosis virus; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... virus; exemption from the requirement of a tolerance. 180.1118 Section 180.1118 Protection of... polyhedrosis virus; exemption from the requirement of a tolerance. An exemption from the requirement of a tolerance is established for the microbial pest control agent Spodoptera exigua nuclear polyhedrosis...

  17. 40 CFR 180.1118 - Spodoptera exigua nuclear polyhedrosis virus; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... virus; exemption from the requirement of a tolerance. 180.1118 Section 180.1118 Protection of... polyhedrosis virus; exemption from the requirement of a tolerance. An exemption from the requirement of a tolerance is established for the microbial pest control agent Spodoptera exigua nuclear polyhedrosis...

  18. 40 CFR 180.1118 - Spodoptera exigua nuclear polyhedrosis virus; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... virus; exemption from the requirement of a tolerance. 180.1118 Section 180.1118 Protection of... polyhedrosis virus; exemption from the requirement of a tolerance. An exemption from the requirement of a tolerance is established for the microbial pest control agent Spodoptera exigua nuclear polyhedrosis...

  19. 40 CFR 180.1118 - Spodoptera exigua nuclear polyhedrosis virus; exemption from the requirement of a tolerance.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... virus; exemption from the requirement of a tolerance. 180.1118 Section 180.1118 Protection of... polyhedrosis virus; exemption from the requirement of a tolerance. An exemption from the requirement of a tolerance is established for the microbial pest control agent Spodoptera exigua nuclear polyhedrosis...

  20. Purification and biochemical characterization of a 70 kDa sericin from tropical tasar silkworm, Antheraea mylitta.

    PubMed

    Dash, Rupesh; Ghosh, Sudip K; Kaplan, David L; Kundu, S C

    2007-05-01

    Sericin isolated from the cocoon of the tropical tasar silkmoth Antheraea mylitta showed three major bands, with the lowest 70 kDa. This band was purified by anion exchange chromatography. Immunoblotting with concanavalin-A suggests a glycoprotein and CD analysis of secondary structure includes beta-sheet. Amino acid analysis shows that the protein is enriched in glycine and serine while the mole percentages of these two amino acids are different from sericin of mulberry silkworm. An anti A. mylitta sericin antibody was able to cross-react with sericin from A. assamensis but not the sericin of Bombyx mori and Philosamia ricini. Immunoblot analysis with proteins isolated from middle silk gland of A. mylitta at different developmental stages of larva showed that the 70 kDa sericin is developmentally regulated. These data extend the range of biochemical features found in this unusual family of proteins and may help in developing an improved understanding of their role in forming environmentally stable fibroin fiber-sericin composite structures (cocoons). PMID:17350301

  1. Crystallization and preliminary X-ray diffraction analysis of a protease inhibitor from the haemolymph of the Indian tasar silkworm Antheraea mylitta

    SciTech Connect

    Roy, Sobhan; Aravind, Penmatsa; Madhurantakam, Chaithanya; Ghosh, Ananta Kumar; Sankaranarayanan, Rajan; Das, Amit Kumar

    2006-07-01

    The crystallization and preliminary X-ray crystallographic analysis of a protease inhibitor from the haemolymph of the Indian tasar silk worm A. mylitta is reported. A protein with inhibitory activity against fungal proteases was purified from the haemolymph of the Indian tasar silkworm Antheraea mylitta and was crystallized using the hanging-drop vapour-diffusion method. Polyethylene glycol 3350 was used as a precipitant. Crystals belonged to space group P6{sub 3}22, with unit-cell parameters a = b = 60.6, c = 85.1 Å. X-ray diffraction data were collected and processed to a maximum resolution of 2.1 Å.

  2. Genetic characterisation of microsporidia infecting Indian tasar silkworm, Antheraea mylitta, using morphology and molecular tools.

    PubMed

    Hassan, Wazid; Surendra Nath, B

    2015-01-01

    The utility of inter simple sequence repeat-PCR (ISSR-PCR) assay in the genetic characterisation and elucidation of the phylogenetic relationship of different microsporidian isolates infecting tropical tasar silkworm, Antheraea mylitta Drury, is demonstrated. A total of 22 different microsporidians collected from the diseased tasar silkworms from Jharkhand state of India were analysed using morphological characters and ISSR-PCR. Observations spores under phase contrast microscope revealed oval to elongate in shape with length ranging from 3.8 μm to 5.1 μm and width from 2.6 μm to 3.3 μm. All the microsporidian isolates except MIJ-1gC showed gonadal infection and transovarial transmission in infected tasar silkworms. Fourteen out of 20 ISSR primers tested generated reproducible profiles and yielded a total of 281 fragments, of which 273 were polymorphic (97%). The degree of banding pattern was used to evaluate genetic distances and for phylogenetic analysis. The results demonstrated that ISSR analysis may be a useful and efficient tool for taxonomical grouping and phylogenetic classification of different microsporidians in general. PMID:26198429

  3. Genome segment 6 of Antheraea mylitta cypovirus encodes a structural protein with ATPase activity

    SciTech Connect

    Chavali, Venkata R.M.; Madhurantakam, Chaithanya; Ghorai, Suvankar; Roy, Sobhan; Das, Amit K.; Ghosh, Ananta K.

    2008-07-20

    The genome segment 6 (S6) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus was converted into cDNA, cloned and sequenced. S6 consisted of 1944 nucleotides with an ORF of 607 amino acids and could encode a protein of 68 kDa, termed P68. Motif scan and molecular docking analysis of P68 showed the presence of two cystathionine beta synthase (CBS) domains and ATP binding sites. The ORF of AmCPV S6 was expressed in E. coli as His-tag fusion protein and polyclonal antibody was raised. Immunoblot analysis of virus infected gut cells and purified polyhedra using raised anti-p68 polyclonal antibody showed that S6 encodes a viral structural protein. Fluorescence and ATPase assay of soluble P68 produced in Sf-9 cells via baculovirus expression system showed its ability to bind and cleave ATP. These results suggest that P68 may bind viral RNA through CBS domains and help in replication and transcription through ATP binding and hydrolysis.

  4. PHYSICAL MAPS OF 'AUTOGRAPHA CALIFORNICA' AND 'RACHIPLUSIA OU' NUCLEAR POLYHEDROSIS VIRUS RECOMBINANTS

    EPA Science Inventory

    TN-368 cells were infected simultaneously with the closely related Autographa californica (AcMNPV) and Rachiplusia ou (RoMNPV) nuclear polyhedrosis viruses. Progeny viral isolates were plaque purified, and their DNAs were analyzed with restriction endonucleases. Of 100 randomly c...

  5. 'SPODOPTERA FRUGIPERDA' NUCLEAR POLYHEDROSIS VIRUS GENOME: PHYSICAL MAPS FOR RESTRICTION ENDONUCLEASES BAMHI AND HINDIII

    EPA Science Inventory

    The physical map for the genome of Spodoptera frugiperda nuclear polyhedrosis virus was constructed for restriction endonucleases BamHI and HindIII. The ordering of the restriction fragments was accomplished by cross-blot hybridization of BamHI, HindIII, and EcoRI fragments. The ...

  6. Inactivation of nuclear polyhedrosis virus (Baculovirus subgroup A) by monochromatic UV radiation

    SciTech Connect

    Griego, V.M.; Martignoni, M.E.; Claycomb, A.E.

    1985-03-01

    Monochromatic radiation at wavelengths of 290, 300, 310, and 320 nm inactivated occluded nuclear polyhedrosis virus of the Douglas-fir tussock moth, Orgyia pseudotsugata. Data indicate that all of the wavelengths are capable of causing virus inactivation; much greater fluences are needed for virus inactivation as the wavelength increases.

  7. AUTOGRAPHA CALIFORNICA NUCLEAR POLYHEDROSIS VIRUS EFFICIENTLY ENTERS BUT DOES NOT REPLICATE IN POIKILOTHERMIC VERTEBRATE CELLS

    EPA Science Inventory

    The host range of the insect virus Autographa californica nuclear polyhedrosis virus (AcMNPV) was examined. AcMNPV could not initiate a productive infection in frog, turtle, trout, or moth cell lines. After exposure to AcMNPV, neither viral DNA nor RNA synthesis could be detected...

  8. Identification of RAPD and SCAR markers associated with yield traits in the Indian tropical tasar silkworm Antheraea mylitta drury.

    PubMed

    Dutta, Suhrid R; Kar, Prasanta K; Srivastava, Ashok K; Sinha, Manoj K; Shankar, Jai; Ghosh, Ananta K

    2012-12-01

    The tropical tasar silkworm, Antheraea mylitta, is a semi-domesticated vanya silk-producing insect of high economic importance. To date, no molecular marker associated with cocoon and shell weights has been identified in this species. In this report, we identified a randomly amplified polymorphic DNA (RAPD) marker and examined its inheritance, and also developed a stable diagnostic sequence-characterized amplified region (SCAR) marker. Silkworms were divided into groups with high (HCSW) and low (LCSW) cocoon and shell weights, and the F(2) progeny of a cross between these two groups were obtained. DNA from these silkworms was screened by PCR using 34 random primers and the resulting RAPD fragments were used for cluster analysis and discriminant function analysis (DFA). The clustering pattern in a UPGMA-based dendogram and DFA clearly distinguished the HCSW and LCSW groups. Multiple regression analysis identified five markers associated with cocoon and shell weights. The marker OPW16(905 bp) showed the most significant association with cocoon and shell weights, and its inheritance was confirmed in F(2) progeny. Cloning and sequencing of this 905 bp fragment showed 88% identity between its 134 nucleotides and the Bmc-1/Yamato-like retroposon of A. mylitta. This marker was further converted into a diagnostic SCAR marker (SCOPW 16(826 bp)). The SCAR marker developed here may be useful in identifying the right parental stock of tasar silk-worms for high cocoon and shell weights in breeding programs designed to enhance the productivity of tasar silk. PMID:23271934

  9. Effects of Substituting Granulin or a Granulin-Polyhedrin Chimera for Polyhedrin on Virion Occlusion and Polyhedral Morphology in Autographa californica Multinucleocapsid Nuclear Polyhedrosis Virus

    PubMed Central

    Eason, Jane E.; Hice, Robert H.; Johnson, Jeffrey J.; Federici, Brian A.

    1998-01-01

    Substitution of granulin from the Trichoplusia ni granulosis virus (TnGV) for polyhedrin of the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) yielded a few very large (2 to 5 μm) cuboidal inclusions in the cytoplasm and nucleus of infected cells. These polyhedra lacked the beveled edges characteristic of wild-type AcMNPV polyhedra, contained fractures, and occluded few virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus and resulted in more structurally uniform cuboidal inclusions in which no virions were observed. A granulin-polyhedrin chimera produced tetrahedral occlusions with more virions than granulin inclusions but many fewer than wild-type polyhedra. Despite the unusual structure of the granulin and granulin-polyhedrin inclusions, they interacted with AcMNPV p10 fibrillar structures and electron-dense spacers that are precursors of the polyhedral calyx. The change in inclusion shape obtained with the granulin-polyhedrin chimera demonstrates that the primary amino acid sequence affects occlusion body shape, but the large cuboidal inclusions formed by granulin indicate that the amino acid sequence is not the only determinant. The failure of granulin or the granulin-polyhedrin chimera to properly occlude AcMNPV virions suggests that specific interactions occur between polyhedrin and other viral proteins which facilitate normal virion occlusion and occlusion body assembly and shape in baculoviruses. PMID:9621097

  10. COMPARISON OF BIOASSAY AND ENZYME-LINKED IMMUNOSORBENT ASSAY FOR QUANTIFICATION OF 'SPODOPTERA FRUGIPERDA' NUCLEAR POLYHEDROSIS VIRUS IN SOIL

    EPA Science Inventory

    Standard curves with known amounts of Spodoptera frugiperda nuclear polyhedrosis virus (NPV) in soil were established with a bioassay and with an enzyme-linked immunosorbent assay (ELISA). The bioassay detected as few as 4 x 10 to the 4th power polyhedral inclusion bodies (PIB)/g...

  11. Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

    PubMed Central

    Smith, Gale E.; Fraser, M. J.; Summers, Max D.

    1983-01-01

    We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus. Images PMID:16789242

  12. Chloroplast and Cytoplasmic Enzymes

    PubMed Central

    Anderson, Louise E.; Pacold, Ivan

    1972-01-01

    Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. The Michaelis constant for fructose-1,6-diphosphate is 19 μM for the chloroplast, 21 μM for the cytoplasmic enzyme, and for sedoheptulose 1,7-diphosphate, 8 μM for the chloroplast enzyme, 18 μM for the cytoplasmic enzyme. Both enzymes are inhibited by d-glyceraldehyde 3-phosphate and by ribulose 1,5-diphosphate. The similarity in the catalytic properties of the isoenzymes suggests that both enzymes have an amphibolic role in carbon metabolism in the green leaf. PMID:16657968

  13. Photoreactivation and ultraviolet-enhanced reactivation of ultraviolet-irradiated nuclear polyhedrosis virus by insect cells.

    PubMed

    Witt, D J

    1984-01-01

    The nuclear polyhedrosis virus (Baculovirus) of Galleria mellonella (Pryalidae: Lepidoptera) was used to investigate the capability of cultured insect cells to repair ultraviolet (UV) induced damage in the viral genome. When assayed by the formation of plaques in the cell line TN-368, the survival of the virus was found to decrease linearly with increased ultraviolet exposure. The infectious capacity of UV-irradiated virions was significantly restored after exposing the TN-368 monolayers to either photoreactivation conditions (white fluorescent and black light) or to UV-enhanced reactivation conditions (far ultraviolet radiation). Using both types of repair sequentially resulted in higher reactivation than when either was used alone. These results indicate that pyrimidine dimers are the major factor responsible for inactivation of this virus by UV radiation but that other photolesions not repairable by photoreactivation partially account for the inactivation of the virus. PMID:6365037

  14. Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system.

    PubMed

    Motohashi, Tomoko; Shimojima, Tsukasa; Fukagawa, Tatsuo; Maenaka, Katsumi; Park, Enoch Y

    2005-01-21

    Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. PMID:15596136

  15. Tissue-specific expression of silkmoth chorion genes in vivo using Bombyx mori nuclear polyhedrosis virus as a transducing vector.

    PubMed Central

    Iatrou, K; Meidinger, R G

    1990-01-01

    A pair of silkmoth chorion chromosomal genes, HcA.12-HcB.12, was inserted into a baculovirus transfer vector, pBmp2, derived from the nuclear polyhedrosis virus of Bombyx mori. This vector, which permits the insertion of foreign genetic material in the vicinity of a mutationally inactivated polyhedrin gene, was used to acquire the corresponding recombinant virus. Injection of mutant silkmoth pupae that lack all Hc chorion genes with the recombinant virus resulted in the infection of all internal organs including follicular tissue. Analysis of RNA from infected tissues has demonstrated that the two chorion genes present in the viral genome are correctly transcribed under the control of their own promoter in follicular cells, the tissue in which chorion genes are normally expressed. The chorion primary transcripts are also correctly processed in the infected follicular cells and yield mature mRNAs indistinguishable from authentic chorion mRNAs present in wild-type follicles. These results demonstrate that recombinant nuclear polyhedrosis viruses can be used as transducing vectors for introducing genetic material of host origin into the cells of the organism and that the transduced genes are transiently expressed in a tissue-specific manner under the control of their resident regulatory sequences. Thus we show the in vivo expression of cloned genes under cellular promoter control in an insect other than Drosophila melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection. Images PMID:2187186

  16. α-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells

    PubMed Central

    Grula, Marjori A.; Buller, Patricia L.; Weaver, Robert F.

    1981-01-01

    [3H]RNA was synthesized in nuclei isolated at various times postinfection from the fat bodies of Heliothis zea larvae infected with H. zea nuclear polyhedrosis virus and from cultured Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus. To detect virus-specific RNA synthesis, the [3H]RNA was hybridized to denatured viral DNA immobilized on nitrocellulose filters. Nuclear polyhedrosis virus-specific RNA synthesis in the infected nuclei isolated from H. zea larval fat bodies and S. frugiperda cells was only inhibited 20 to 25% by concentrations of α-amanitin sufficient to inhibit the host RNA polymerase II. In addition, a productive nuclear polyhedrosis virus infection was obtained in S. frugiperda cells grown in the presence of an α-amanitin concentration that inhibited 90% of the cellular RNA polymerase II activity. The cellular RNA polymerase II enzyme remained sensitive to α-amanitin during infection, and there was no evidence that a virus-coded, α-amanitin-resistant enzyme was synthesized after the onset of infection. The data suggest that the bulk of nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei is transcribed by an enzyme other than the host RNA polymerase II. PMID:16789208

  17. Cytoplasmic Z-RNA

    SciTech Connect

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-09-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.

  18. Semipermissive replication of a nuclear polyhedrosis virus of Autographa californica in a gypsy moth cell line

    SciTech Connect

    McClintock, J.T.; Dougherty, E.M.; Weiner, R.M.

    1986-01-01

    Several gypsy moth cell lines have been previously described as nonpermissive for the multiple-embedded nuclear polyhedrosis virus of Autographa californica (AcMNPV). In this report, the authors demonstrate the semipermissive infection of a gypsy moth cell line, IPLB-LD-652Y, with AcMNPV. IPLB-LD-652Y cells infected with AcMNPV produced classic cytopathic effects but failed to yield infectious progeny virus. Results of experiments employing DNA-DNA dot hybridization suggested that AcMNPV DNA synthesis was initiated from 8 to 12 h postinfection (p.i.), continued at a maximum rate from 12 to 20 h p.i., and declined from 20 to 36 h p.i. The rate of AcMNPV DNA synthesis approximated that observed in the permissive TN-368 cell line. AcMNPV-infected IPLB-LD-652Y cells, pulse-labeled with (/sup 35/S)methionine at various time intervals p.i. and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed four virus-induced proteins, one novel to the semipermissive system and three early ..cap alpha.. proteins, synthesized from 1 to 20 h p.i. Thereafter, both host and viral protein synthesis was completely suppressed. These results suggest that AcMNPV adsorbed, penetrated, and initiated limited macromolecular synthesis in the semipermissive gypsy moth cell line. However, the infection cycle was restricted during the early phase of AcMNPV replication.

  19. Characterization of a Nuclear Polyhedrosis Virus Isolated from Diseased Gonometa podocarpi (Lepidoptera:Lasiocampidae)

    PubMed Central

    Moore, Norman F.; Armstrong, Terry; Green, Bernadette; Brown, David; Hibbin, Jill; Kelly, David C.; Tinsley, Thomas W.; Aloo, Theresa C.

    1981-01-01

    Gonometa podocarpi is an important pest of several species of pine in East Africa, and large numbers of trees in plantations in Kenya were partially or completely defoliated by the larval stage of this insect. After the infestation in the Mt. Elgon region, large numbers of dead and moribund larvae were found on the ground. Examination of extracts of these larvae demonstrated the presence of an occluded virus. Electron microscopy of purified sectioned polyhedra demonstrated the presence of virus particles containing from 1 to 12 nucleocapsids. Purification of virus particles from polyhedra was accomplished by using alkali solubilization and sucrose gradient centrifugation. Virus particles contained 15 proteins as determined by polyacrylamide gel electrophoresis. Detergent solubilization of the virus particles released polyhedra containing one major structural protein. Electron microscopy of purified virus particles and nucleocapsids demonstrated them to be similar in structure to previously recorded nuclear polyhedrosis viruses. The viral deoxyribonucleic acid was extracted and spread for electron microscopy and was determined to have a size of approximately 80 × 106 daltons. Images PMID:16345830

  20. Data for increase of Lymantria dispar male survival after topical application of single-stranded RING domain fragment of IAP-3 gene of its nuclear polyhedrosis virus

    PubMed Central

    Oberemok, Volodymyr V.; Laikova, Kateryna V.; Zaitsev, Aleksei S.; Gushchin, Vladimir A.; Skorokhod, Oleksii A.

    2016-01-01

    This data article is related to the research article entitled “The RING for gypsy moth control: topical application of fragment of its nuclear polyhedrosis virus anti-apoptosis gene as insecticide” [1]. This article reports on significantly higher survival of gypsy moth Lymantria dispar male individuals in response to topical application of single-stranded DNA, based on RING (really interesting new gene) domain fragment of LdMNPV (L. dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene and acted as DNA insecticide. PMID:27054151

  1. Data for increase of Lymantria dispar male survival after topical application of single-stranded RING domain fragment of IAP-3 gene of its nuclear polyhedrosis virus.

    PubMed

    Oberemok, Volodymyr V; Laikova, Kateryna V; Zaitsev, Aleksei S; Gushchin, Vladimir A; Skorokhod, Oleksii A

    2016-06-01

    This data article is related to the research article entitled "The RING for gypsy moth control: topical application of fragment of its nuclear polyhedrosis virus anti-apoptosis gene as insecticide" [1]. This article reports on significantly higher survival of gypsy moth Lymantria dispar male individuals in response to topical application of single-stranded DNA, based on RING (really interesting new gene) domain fragment of LdMNPV (L. dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene and acted as DNA insecticide. PMID:27054151

  2. Genetic variability and genetic structure of wild and semi-domestic populations of tasar silkworm (Antheraea mylitta ) ecorace Daba as revealed through ISSR markers.

    PubMed

    Kar, P K; Vijayan, K; Mohandas, T P; Nair, C V; Saratchandra, B; Thangavelu, K

    2005-11-01

    The genetic diversity in the wild and semi-domestic populations of Daba ecorace of Antheraea mylitta was studied to ascertain the distribution of variability within and among populations of semi-domestic bivoltine (DB), trivoltine (DT) and nature grown wild populations (DN) with inter-simple sequence repeat (ISSR) markers. A total of 138 markers were produced among 56 individuals of the three populations, of which 98% were polymorphic. For the individual populations, the percentage polymorphism was 58.69, 52.9 and 77.54 for DB, DT and DN, respectively. Average number of observed (1.791+/- 0.408) and effective alleles (1.389+/-0.348) was also high in the wild populations in comparison to the bivoltine and trivoltine semi-domestic populations. Genetic diversity (H(t)) in DB, DT and DN was 0.180+/- 0.033, 0.153+/- 0.032 and 0.235+/- 0.033, respectively and within-population genetic diversity (H(s)) ranged from 0.166 to 0.259 with a mean of 0.189. Mean gene differentiation (G(ST)) was found to be 0.25. Shanon's diversity index was 0.278, 0.237 and 0.361 for DB, DT and DN and overall it was 0.391. Gene flow (N(m)) among the populations was 1.509. The dendrogram produced by UPGMA with Dice's genetic distance matrices resulted in the formation of three major clusters separating the three populations. Considerable intra- and inter-population variability is found in all three populations. The population structure analysis further suggests that the semi-domestic populations of Daba ecorace are at the threshold of differentiating themselves. The high genetic variability present within wild Daba population of A. mylitta is of much importance for conservation as well as utilization in systematic breeding program. PMID:16247690

  3. DNA hybridization assay for detection of gypsy moth nuclear polyhedrosis virus in infected gypsy moth (Lymantria dispar L. ) larvae

    SciTech Connect

    Keating, S.T.; Burand, J.P.; Elkinton, J.S. )

    1989-11-01

    Radiolabeled Lymantria dispar nuclear polyhedrosis virus DNA probes were used in a DNA hybridization assay to detect the presence of viral DNA in extracts from infected larvae. Total DNA was extracted from larvae, bound to nitrocellulose filters, and assayed for the presence of viral DNA by two methods: slot-blot vacuum filtration and whole-larval squashes. The hybridization results were closely correlated with mortality observed in reared larvae. Hybridization of squashes of larvae frozen 4 days after receiving the above virus treatments also produced accurate measures of the incidence of virus infection.

  4. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F.W.; Rosenberg, A.H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest. 1 fig.

  5. Cytoplasmic bacteriophage display system

    DOEpatents

    Studier, F. William; Rosenberg, Alan H.

    1998-06-16

    Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest.

  6. A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

    PubMed Central

    Ohkawa, T; Majima, K; Maeda, S

    1994-01-01

    Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997

  7. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

    SciTech Connect

    Kitajima, Masayuki; Hamazaki, Hiroyuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi . E-mail: hiroshi.takaku@it-chiba.ac.jp

    2006-05-05

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.

  8. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells.

    PubMed

    Kitajima, Masayuki; Hamazaki, Hiroyuki; Miyano-Kurosaki, Naoko; Takaku, Hiroshi

    2006-05-01

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type. PMID:16545777

  9. A mechanism for negative gene regulation in Autographa californica multinucleocapsid nuclear polyhedrosis virus

    USGS Publications Warehouse

    Leisy, D.J.; Rasmussen, C.; Owusu, E.O.; Rohrmann, G.F.

    1997-01-01

    The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) ie-1 gene product (IE-1) is thought to play a central role in stimulating early viral transcription. IE-1 has been demonstrated to activate several early viral gene promoters and to negatively regulate the promoters of two other AcMNPV regulatory genes, ie-0 and ie-2. Our results indicate that IE-1 negatively regulates the expression of certain genes by binding directly, or as part of a complex, to promoter regions containing a specific IE-1-binding motif (5'-ACBYGTAA-3') near their mRNA start sites. The IE-1 binding motif was also found within the palindromic sequences of AcMNPV homologous repeat (hr) regions that have been shown to bind IE-1. The role of this IE-1 binding motif in the regulation of the ie-2 and pe-38 promoters was examined by introducing mutations in these promoters in which the central 6 bp were replaced with Bg/II sites. GUS reporter constructs containing ie-2 and pe-38 promoter fragments with and without these specific mutations were cotransfected into Sf9 cells with various amounts of an ie-1-containing plasmid (ple-1). Comparisons of GUS expression produced by the mutant and wild-type constructs demonstrated that the IE-1 binding motif mediated a significant decrease in expression from the ie-2 and pe-38 promoters in response to increasing pIe-1 concentrations. Electrophoretic mobility shift assays with pIe-1-transfected cell extracts and supershift assays with IE-1- specific antiserum demonstrated that IE-1 binds to promoter fragments containing the IE-1 binding motif but does not bind to promoter fragments lacking this motif.

  10. SELECTION KINETICS DURING SERIAL CELL CULTURE PASSAGE OF MIXTURES OF WILD TYPE AUTOGRAPHA CALIFORNICA NUCLEAR POLYHEDROSIS VIRUS AND ITS RECOMBINANT AC360-B-GAL

    EPA Science Inventory

    Detailed analysis of the selection process in serial co-infections of cell cultures by wild type Autographa californica nuclear polyhedrosis virus, AcNPV/E2, and Ac36O-B-gal, a "genetically engineered" strain, shows that the unaltered strain was clearly dominant even when it bega...

  11. Risk Assessment Studies: Detailed Host Range Testing of Wild-Type Cabbage Moth, Mamestra brassicae (Lepidoptera: Noctuidae), Nuclear Polyhedrosis Virus

    PubMed Central

    Doyle, Cathy J.; Hirst, Mark L.; Cory, Jenny S.; Entwistle, Philip F.

    1990-01-01

    The host range of a multiply enveloped nuclear polyhedrosis virus (NPV) (Baculoviridae) isolated from the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae), was determined by challenging a wide range of insect species with high (106 polyhedral inclusion bodies) and low (103 polyhedral inclusion bodies) doses of the virus. The identity of the progeny virus was confirmed by dot blotting. Analysis of 50% lethal dose was carried out on selected species, and the progeny virus was identified by using restriction enzyme analysis and Southern blotting. Other than the Lepidoptera, none of the species tested was susceptible to M. brassicae NPV. Within the Lepidoptera, M. brassicae NPV was infective to members of four families (Noctuidae, Geometridae, Yponomeutidae, and Nymphalidae). Of 66 lepidopterous species tested, M. brassicae NPV was cross-infective to 32 of them; however, 91% of the susceptible species were in the Noctuidae. The relevance of host range data in risk assessment studies is discussed. Images PMID:16348279

  12. Effect of aluminum chloride and zinc sulfate on Autographa california nuclear polyhedrosis virus (ACNPV) replication in cell culture.

    PubMed

    Weiss, S A; Smith, G C; Vaughn, J L; Dougherty, E M; Tompkins, G J

    1982-11-01

    When IPL-SF-21AE III continuous insect cell line was grown and maintained in IPL-41 insect cell culture medium supplemented with 16 microM of AlCl3 or 0.24 microM of ZnSO4 . 7H2O, or both metallic salts, and then infected with Autographa california nuclear polyhedrosis virus, virus replication was increased significantly. The yield of polyhedral inclusion bodies (PIB) was enhanced up to 121%. Synthesis of cell-free nonoccluded virus was increased to 365% when infectivity was assayed by the plaque method. Newly applied electron microscopic quantitation and stereological techniques also revealed a significant increase in virus particles (VP) and in amount and size of PIB as well as number of VP per PIB. PMID:6759370

  13. Oxidative Damaged Products, Level of Hydrogen Peroxide, and Antioxidant Protection in Diapausing Pupa of Tasar Silk Worm, Antheraea mylitta: A Comparative Study in Two Voltine Groups

    PubMed Central

    Sahoo, Alpana; Dandapat, Jagneshwar; Samanta, Luna

    2015-01-01

    The present study demonstrates tissue-specific (hemolymph and fat body) and inter-voltine [bivoltine (BV) and trivoltine (TV)] differences in oxidatively damaged products, H2O2 content, and the relative level of antioxidant protection in the diapausing pupae of Antheraea mylitta. Results suggest that fat body (FB) of both the voltine groups has oxidative predominance, as evident from the high value of lipid peroxidation and H2O2 content, despite better enzymatic defenses in comparison to hemolymph (HL). This may be attributed to the higher metabolic rate of the tissue concerned, concomitant with high lipid content and abundance of polyunsaturated fatty acids (PUFA). Nondetectable catalase activity in the pupal hemolymph of both strains apparently suggests an additional mechanism for H2O2 metabolism in the tissue. Inter-voltine comparison of the oxidative stress indices and antioxidant defense potential revealed that the TV group has a higher oxidative burden, lower activities for the antioxidant enzymes, and compensatory nonenzymatic protection from reduced glutathione and ascorbic acid. PMID:26816485

  14. Oxidative Damaged Products, Level of Hydrogen Peroxide, and Antioxidant Protection in Diapausing Pupa of Tasar Silk Worm, Antheraea mylitta: A Comparative Study in Two Voltine Groups.

    PubMed

    Sahoo, Alpana; Dandapat, Jagneshwar; Samanta, Luna

    2015-01-01

    The present study demonstrates tissue-specific (hemolymph and fat body) and inter-voltine [bivoltine (BV) and trivoltine (TV)] differences in oxidatively damaged products, H2O2 content, and the relative level of antioxidant protection in the diapausing pupae of Antheraea mylitta. Results suggest that fat body (FB) of both the voltine groups has oxidative predominance, as evident from the high value of lipid peroxidation and H2O2 content, despite better enzymatic defenses in comparison to hemolymph (HL). This may be attributed to the higher metabolic rate of the tissue concerned, concomitant with high lipid content and abundance of polyunsaturated fatty acids (PUFA). Nondetectable catalase activity in the pupal hemolymph of both strains apparently suggests an additional mechanism for H2O2 metabolism in the tissue. Inter-voltine comparison of the oxidative stress indices and antioxidant defense potential revealed that the TV group has a higher oxidative burden, lower activities for the antioxidant enzymes, and compensatory nonenzymatic protection from reduced glutathione and ascorbic acid. PMID:26816485

  15. Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia ni: comparative neutralization comparative infectivity, and in vitro growth studies.

    PubMed Central

    Volkman, L E; Summers, M D; Hsieh, C H

    1976-01-01

    Nuclear polyhedrosis virus infections of lepidopteran cells often result in the production of both occluded and nonoccluded virus. The characterization of these two different forms has been the subject of several papers. We have divided the nonoccluded virus (NOV) category further into plasma membrane-budded non-occluded virus (PMB-NOV), intracellular NOV, and hemolymph-derived NOV, and have done additional studies investigating the differences between these nonoccluded forms and the alkali-liberated forms from occlusions of the nuclear polyhedrosis viruses of Autographa californica and Rachiplusa ou. The methods used to discern differences and similarities among the forms were serological, biochemical, and visual, all related to their biological acitivity. Neutralization studies revealed that alkali-liberated virus and PMB-NOV had both similar and different antigens. Antisera raised against alkali-liberated virus from occlusions neutralized the alkali-liberated form of the virus, but did not neutralize the intracellular or extracellular nonoccluded forms. Antisera raised against the TN-368-13 PMB-NOV, however, neutralized the alkali-liberated forms as well as all forms of the NOV. Adsorption of this antisera with alkali-liberated virus did not diminish the neutralization titer against the nonoccluded forms, thus confirming the antigenic differences between the alkali-liberated and nonoccluded forms of the virus. Physical-infectious particle ratio calculations indicated that the PMB-NOV of Autographa californica are about 1,900-fold more infectious than the single-nucleocapsid-per-envelope alkali-liberated particles and about 1,700-fold more infectious than the multiple-nucleocapsid-per-envelope particles, as assayed in vitro. In addition, a study of viral growth kinetics monitored concurrently with the appearance of polyhedra showed that PMB-NOV production is shut down with the onset of polyhedron formation. PMID:787558

  16. Identification of three late expression factor genes within the 33.8- to 43.4-map-unit region of Autographa californica nuclear polyhedrosis virus.

    PubMed Central

    Lu, A; Miller, L K

    1994-01-01

    A transient transactivation assay system was used in combination with an overlapping Autographa californica nuclear polyhedrosis virus clone library to identify genes involved in late and very late baculovirus gene expression. We have identified three genes within the 33.8- to 43.4-map-unit region of the A. californica nuclear polyhedrosis virus genome which contribute to expression from promoters of the vp39 major capsid protein and polyhedrin genes. One of these three genes corresponds to the previously identified DNA polymerase gene, while the other two genes encode previously unidentified polypeptides of 59,418 and 8,706 Da. None of these genes were required for expression from the early etl promoter. Images PMID:8084003

  17. Structural studies on the polyhedral inclusion bodies, virions, and DNA of the nuclear polyhedrosis virus of the cotton bollworm Heliothis zea.

    PubMed Central

    Scharnhorst, D W; Saving, K L; Vuturo, S B; Cooke, P H; Weaver, R F

    1977-01-01

    The polyhedral inclusion body of the cotton bollworm nuclear polyhedrosis virus contains virions occluded in an orthogonal crystalline matrix. The virions appear as rods or, more frequently, as oval structures that form upon bending of the nucleocapsid within the viral membrane. The nucleocapsid consists at least of DNA surrounded by a capsid composed of subunits, possibly helically arranged. The viral DNA is circular and supercoiled. It is heterogenous in size with contour lengths ranging from 15 to 45 mum. Images PMID:319251

  18. Splicing is required for transactivation by the immediate early gene 1 of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.

    PubMed

    Pearson, M N; Rohrmann, G F

    1997-08-18

    A region of the Lymantria disper multinucleocapsid nuclear polyhedrosis virus (LdMNPV) genome containing the homolog of the baculovirus ie-1 gene was identified using a series of overlapping cosmids and individual plasmids in a transient transcriptional expression assay. Sequence analysis of the active region identified two ORFs, one of which is 32% identical to AcMNPV ORF141 (ie-0) and contains a putative splice donor site and the other of which is 29% identical to AcMNPV ie-1 and contains a highly conserved splice acceptor consensus sequences. Plasmids containing the LdMNPV ORF141 and ie-1 regions were able to stimulate expression of a GUS reporter gene, while plasmids containing the ie-1 region alone were inactive, suggesting that only the spliced, IE-0 form of the gene product is an active transactivator. Primer extension analysis confirmed the presence of spliced ie-0 mRNA transcripts starting at 6 hr and continuing throughout the time course of viral infection of the L dispar cell line Ld652Y. Using a plasmid containing the ie-0 spliced form of the gene as a transactivator, hr4, one of the eight homologous regions of LdMNPV, was shown to act as a transcriptional enhancer. In contrast, a reporter plasmid containing the AcMNPV hr5 enhancer did not show increased activity when cotransfected with LdMNPV ie-0, suggesting that these enhancer sequences are viral specific. In a transient replication assay system. LdMNPV ie-0 acted as an essential replication gene, but LdMNPV ie-1 was inactive. These results indicate that splicing is required to obtain an active gene product in LdMNPV in the Ld652Y cell line. PMID:9300047

  19. An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs.

    PubMed Central

    Birnbaum, M J; Clem, R J; Miller, L K

    1994-01-01

    Two different baculovirus genes are known to be able to block apoptosis triggered upon infection of Spodoptera frugiperda cells with p35 mutants of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV):p35 (P35-encoding gene) of AcMNPV (R. J. Clem, M. Fechheimer, and L. K. Miller, Science 254:1388-1390, 1991) and iap (inhibitor of apoptosis gene) of Cydia pomonella granulosis virus (CpGV) (N. E. Crook, R. J. Clem, and L. K. Miller, J. Virol. 67:2168-2174, 1993). Using a genetic complementation assay to identify additional genes which inhibit apoptosis during infection with a p35 mutant, we have isolated a gene from Orgyia pseudotsugata NPV (OpMNPV) that was able to functionally substitute for AcMNPV p35. The nucleotide sequence of this gene, Op-iap, predicted a 30-kDa polypeptide product with approximately 58% amino acid sequence identity to the product of CpGV iap, Cp-IAP. Like Cp-IAP, the predicted product of Op-iap has a carboxy-terminal C3HC4 zinc finger-like motif. In addition, a pair of additional cysteine/histidine motifs were found in the N-terminal regions of both polypeptide sequences. Recombinant p35 mutant viruses carrying either Op-iap or Cp-iap appeared to have a normal phenotype in S. frugiperda cells. Thus, Cp-IAP and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism which may involve direct interaction with DNA. Images PMID:8139034

  20. An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs.

    PubMed

    Birnbaum, M J; Clem, R J; Miller, L K

    1994-04-01

    Two different baculovirus genes are known to be able to block apoptosis triggered upon infection of Spodoptera frugiperda cells with p35 mutants of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV):p35 (P35-encoding gene) of AcMNPV (R. J. Clem, M. Fechheimer, and L. K. Miller, Science 254:1388-1390, 1991) and iap (inhibitor of apoptosis gene) of Cydia pomonella granulosis virus (CpGV) (N. E. Crook, R. J. Clem, and L. K. Miller, J. Virol. 67:2168-2174, 1993). Using a genetic complementation assay to identify additional genes which inhibit apoptosis during infection with a p35 mutant, we have isolated a gene from Orgyia pseudotsugata NPV (OpMNPV) that was able to functionally substitute for AcMNPV p35. The nucleotide sequence of this gene, Op-iap, predicted a 30-kDa polypeptide product with approximately 58% amino acid sequence identity to the product of CpGV iap, Cp-IAP. Like Cp-IAP, the predicted product of Op-iap has a carboxy-terminal C3HC4 zinc finger-like motif. In addition, a pair of additional cysteine/histidine motifs were found in the N-terminal regions of both polypeptide sequences. Recombinant p35 mutant viruses carrying either Op-iap or Cp-iap appeared to have a normal phenotype in S. frugiperda cells. Thus, Cp-IAP and Op-IAP appear to be functionally analogous to P35 but are likely to block apoptosis by a different mechanism which may involve direct interaction with DNA. PMID:8139034

  1. The role of cytochrome c on apoptosis induced by Anagrapha falcifera multiple nuclear polyhedrosis virus in insect Spodoptera litura cells.

    PubMed

    Liu, Kaiyu; Shu, Duanyang; Song, Na; Gai, Zhongchao; Yuan, Yuan; Li, Juan; Li, Min; Guo, Shuying; Peng, Jianxin; Hong, Huazhu

    2012-01-01

    There are conflicting reports on the role of cytochrome c during insect apoptosis. Our previous studies have showed that cytochrome c released from the mitochondria was an early event by western blot analysis and caspase-3 activation was closely related to cytochrome c release during apoptosis induced by baculovirus in Spodoptera litura cells (Sl-1 cell line). In the present study, alteration in mitochondrial morphology was observed by transmission electron microscopy, and cytochrome c release from mitochondria in apoptotic Sl-1 cells induced with Anagrapha falcifera multiple nuclear polyhedrosis virus (AfMNPV) has further been confirmed by immunofluoresence staining protocol, suggesting that structural disruption of mitochondria and the release of cytochrome c are important events during Lepidoptera insect cell apoptosis. We also used Sl-1 cell-free extract system and the technique of RNA interference to further investigate the role of cytochrome c in apoptotic Sl-1 cells induced by AfMNPV. Caspase-3 activity in cell-free extracts supplemented with exogenous cytochrome c was determined and showed an increase with the extension of incubation time. DsRNA-mediated silencing of cytochrome c resulted in the inhibition of apoptosis and protected the cells from AfMNPV-induced cell death. Silencing of expression of cytochrome c had a remarkable effect on pro-caspase-3 and pro-caspase-9 activation and resulted in the reduction of caspase-3 and caspase-9 activity in Sl-1 cells undergoing apoptosis. Caspase-9 inhibitor could inhibit activation of pro-caspase-3, and the inhibition of the function of Apaf-1 with FSBA blocked apoptosis, hinting that Apaf-1 could be involved in Sl-1 cell apoptosis induced by AfMNPV. Taken together, these results strongly demonstrate that cytochrome c plays an important role in apoptotic signaling pathways in Lepidopteran insect cells. PMID:22952575

  2. Isolation of Cytoplasmic Enzymes from Pollen 1

    PubMed Central

    Weeden, Norman F.; Gottlieb, Leslie D.

    1980-01-01

    The cytoplasmic isozyme of many cytoplasmic-organelle isozyme pairs, as well as other cytoplasmic enzymes in plants, can be readily obtained from pollen by soaking it in an appropriate buffer for 4 hours. Enzymes localized in subcellular organelles appear not to be released during the soaking period, although they are released if the pollen is crushed. The technique is a useful initial step in studies of subcellular localization of enzymes or for obtaining small quantities of cytoplasmic enzymes free of organellar contaminants. Images PMID:16661444

  3. Measurement of Cytoplasmic Streaming in Drosophila Melanogaster

    NASA Astrophysics Data System (ADS)

    Ganguly, Sujoy; Williams, Lucy; Palacios, Isabel; Goldstein, Raymond

    2010-11-01

    During stage 9 of Drosophila melanogastor oogenesis flow of the oocyte cytoplasm, driven by kinesin 1 motor protein is observed. This cytoplasmic streaming is analyzed by PIV in both wild type and kinesin light chain mutants, revealing striking statistical differences. Further measurements of the rheology of the oocyte allow for estimations of the mechanical energy needed to generate the observed flows.

  4. Cytoplasmic Streaming - Skylab Student Experiment ED-63

    NASA Technical Reports Server (NTRS)

    1973-01-01

    This chart describes the Skylab student experiment (ED-63), Cytoplasmic Streaming, proposed by Cheryl A. Peitz of Arapahoe High School, Littleton, Colorado. Experiment ED-63 was to observe the effect of zero-gravity on cytoplasmic streaming in the aquatic plant named Elodea, commonly called water weed or water thyme. The phenomenon of cytoplasmic streaming is not well understood, but it is recognized as the circulation mechanism of the internal materials or cytoplasm of a cell. Cytoplasm is a gelatinous substance that has the ability to change its viscosity and flow, carrying various cell materials with it. The activity can be stimulated by sunlight or heat. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

  5. Single-stranded DNA fragments of insect-specific nuclear polyhedrosis virus act as selective DNA insecticides for gypsy moth control.

    PubMed

    Oberemok, Volodymyr V; Skorokhod, Oleksii A

    2014-07-01

    This paper focuses on the DNA insecticides as a novel preparation against gypsy moth (Lymantria dispar) based on DNA fragments of the anti-apoptotic gene of its nuclear polyhedrosis virus. It was found that the external application of a solution with two single-stranded DNA fragments from BIR and RING domains of LdMNPV (L.dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene induces a significantly higher mortality of gypsy moth caterpillars in comparison with the application of the control solutions. This effect does not depend on the infection of caterpillars with LdMNPV. The results also show that DNA insecticides based on LdMNPV IAP-3 gene fragments can be selective in action, and at least are not harmful to tobacco hornworm (Manduca sexta) and black cutworm (Agrotis ipsilon). Part of the gypsy moth genome cloned with the fragments of BIR and RING domains of LdMNPV IAP-3 gene as primers, has an overlap with the corresponding part of the LdMNPV IAP-3 gene and L.dispar IAP-1 mRNA for an inhibitor of apoptosis protein with the high cover by query, allows assuming that we cloned a part of gypsy moth anti-apoptosis gene. This finding gives the grounding that proposed here DNA insecticides might act through the blocking of the mechanisms involved in post transcriptional expression of insect anti-apoptosis genes. The results show the insecticidal potential of the viral genome fragments that can be used to create safe and relatively fast-acting DNA insecticides to control the quantity of gypsy moth populations, important task for forestry and agriculture. PMID:25052520

  6. Deep cytoplasmic rearrangements in ventralized Xenopus embryos

    NASA Technical Reports Server (NTRS)

    Brown, E. E.; Denegre, J. M.; Danilchik, M. V.

    1993-01-01

    Following fertilization in Xenopus, dramatic rearrangements of the egg cytoplasm relocalize maternally synthesized egg components. During the first cell cycle the vegetal yolk mass rotates relative to the egg surface, toward the sperm entry point (SEP) (J. P. Vincent, G. F. Oster, and J. C. Gerhart, 1986, Dev. Biol. 113, 484-500), while concomitant deep cytoplasmic rearrangements occur in the animal hemisphere (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). In this paper we examine the role of vegetal yolk mass rotation in producing the animal cytoplasmic rearrangements. We inhibited rotation by uv-irradiating embryos during the first cell cycle, a treatment that yields an extremely ventralized phenotype. Both uv-irradiated embryos and unirradiated control embryos show cytoplasmic rearrangements in the animal hemisphere during the first cell cycle. Cytoplasmic rearrangements on the SEP side of the embryo associated with the path of the sperm pronucleus, plus a swirl on the anti-SEP (dorsal) side, are seen, whether or not yolk mass rotation has occurred. This result suggests a role for the expanding sperm aster in directing animal hemisphere cytoplasmic movements. In unirradiated control embryos the anti-SEP (dorsal) swirl is larger than that in uv-irradiated embryos and often extends into the vegetal hemisphere, consistent with the animal cytoplasm having been pulled dorsally and vegetally by the sliding vegetal yolk mass. Thus the yolk mass rotation may normally enhance the dorsalward cytoplasmic movement, begun by the sperm aster, enough to induce normal axis formation. We extended our observations of unirradiated control and uv-irradiated embryos through early cleavages. The vegetal extent of the anti-SEP (dorsal) swirl pattern seen in control embryos persists through the early cleavage period, such that labeled animal cytoplasm extends deep into dorsal third-tier blastomeres at the 32-cell stage. Significantly, in uv-irradiated embryos

  7. Cytoplasmic rearrangements associated with amphibian egg symmetrization

    NASA Technical Reports Server (NTRS)

    Malacinski, G. M.

    1984-01-01

    Cytoplasmic rearrangements which follow fertilization were mentioned in normal and inverted eggs. A set of yolk compartments was resolved by cytological analyses of both normally oriented and inverted eggs. Those compartments were characterized by their yolk platelet compositions and movement during egg inversion. It is found that during egg inversion the yolk compartments shift minor cytoplasmic compartments which line the egg cortex. Those yolk mass shifts occurred only after the inverted egg was activated. The direction of shift of the major yolk components, rather than the sperm entrance site, determines the dorsal/ventral polarity of the inverted egg. Among different spawnings the rate of shift varied. Eggs that displayed the fastest rate of shift exhibited the highest frequency of developmental abnormalities during organogenesis. Interpretation of novel observations on cytoplasmic organization provide criticism of some earlier models. A new density compartment model is presented as a coherent way to view the organization of the egg cytoplasm and the development of bilateral symmetry.

  8. The mechanics of motility in dissociated cytoplasm.

    PubMed Central

    Dembo, M

    1986-01-01

    We stimulate the dynamical behavior of dissociated cytoplasm using the Reactive Flow Model (Dembo, M., and F. Harlow, 1986, Biophys. J., 50:109-121). We find that for the most part the predicted dynamical behavior of the cytoplasm is governed by three nondimensional numbers. Several other nondimensional parameters, the initial conditions, and boundary conditions are found to have lesser effects. Of the three major nondimensional parameters, one (D#) controls the percentage of ectoplasm, the second (C#) controls the sharpness of the endoplasm-ectoplasm boundary, and the third (R#) controls the topological complexity of the endoplasm-ectoplasm distribution. If R# is very small, then the cytoplasm contracts into a single uniform mass, and there is no bulk streaming. If R# is very large, then the cytoplasmic mass breaks up into a number of clumps scattered throughout the available volume. Between these clumps the solution undergoes turbulent or chaotic patterns of streaming. Intermediate values of R# can be found such that the mass of cytoplasm remains connected and yet undergoes coherent modes of motility similar to flares (Taylor, D.L., J.S. Condeelis, P.L. Moore, and R.D. Allen, 1973, J. Cell Biol., 59:378-394) and rosettes (Kuroda, K., 1979, Cell Motility: Molecules and Organization, 347-362). Images FIGURE 1 FIGURE 1B FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 PMID:3801576

  9. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. PMID:24630119

  10. Consequences of Cytoplasmic Irradiation: Studies from Microbeam

    PubMed Central

    Zhou, Hongning; Hong, Mei; Chai, Yunfei; Hei, Tom K.

    2013-01-01

    The prevailing dogma for radiation biology is that genotoxic effects of ionizing radiation such as mutations and carcinogenesis are attributed mainly to direct damage to the nucleus. However, with the development of microbeam that can target precise positions inside the cells, accumulating evidences have shown that energy deposit by radiation in nuclear DNA is not required to trigger the damage, extra-nuclear or extra-cellular radiation could induce the similar biological effects as well. This review will summarize the biological responses after cytoplasm irradiated by microbeam, and the possible mechanisms involved in cytoplasmic irradiation. PMID:19346686

  11. How crowded is the prokaryotic cytoplasm?

    PubMed

    Spitzer, Jan; Poolman, Bert

    2013-07-11

    We consider biomacromolecular crowding within the cytoplasm of prokaryotic cells as a two-phase system of 'supercrowded' cytogel and 'dilute' cytosol; we simplify and quantify this model for a coccoid cell over a wide range of biomacromolecular crowding. The key result shows that the supercrowded cytogel extends the vectorial character of the plasma membrane deeper into the cytoplasm by about 20-70 nm. We discuss useful physiological insights that this model gives into the functioning of a prokaryotic cell on the micrometer scale. PMID:23735698

  12. Detection of cytoplasmic glycosylation associated with hydroxyproline.

    PubMed

    West, Christopher M; van der Wel, Hanke; Blader, Ira J

    2006-01-01

    A special class of glycosylation occurs on a proline residue of the cytoplasmic/nuclear protein Skp1 in the social amoeba Dictyostelium. For this glycosylation to occur, the proline must first be hydroxylated by the action of a soluble prolyl 4-hydroxylase acting on the protein. Cytoplasmic prolyl 4-hydroxylases are dioxygen-dependent enzymes that have low affinity for their O2 substrate and, therefore, have been implicated in O2-sensing in Dictyostelium, as well as in vertebrates and invertebrates. The sugar-hydroxyproline linkage has low abundance, is resistant to alkali cleavage and known glycosidases, and does not bind known lectins. However, initial screens for this modification can be made by assessing changes in electrophoretic mobility of candidate proteins after treatment of cells with prolyl hydroxylase inhibitors, and/or by metabolic labeling with [3H]sugar precursors. In addition, cytoplasmic hydroxylation/glycosylation can be assessed by assaying for cytoplasmic glycosyltransferases. Here we describe these methods and examples of their use in analyzing Skp1 glycosylation in Dictyostelium and the apicomplexan Toxoplasma gondii, the causative agent of toxoplasmosis in humans. PMID:17132515

  13. CNS Myelination Requires Cytoplasmic Dynein Function

    PubMed Central

    Yang, Michele L.; Shin, Jimann; Kearns, Christina A.; Langworthy, Melissa M.; Snell, Heather; Walker, Macie B.; Appel, Bruce

    2014-01-01

    Background Cytoplasmic dynein provides the main motor force for minus-end-directed transport of cargo on microtubules. Within the vertebrate central nervous system (CNS), proliferation, neuronal migration and retrograde axon transport are among the cellular functions known to require dynein. Accordingly, mutations of DYNC1H1, which encodes the heavy chain subunit of cytoplasmic dynein, have been linked to developmental brain malformations and axonal pathologies. Oligodendrocytes, the myelinating glial cell type of the CNS, migrate from their origins to their target axons and subsequently extend multiple long processes that ensheath axons with specialized insulating membrane. These processes are filled with microtubules, which facilitate molecular transport of myelin components. However, whether oligodendrocytes require cytoplasmic dynein to ensheath axons with myelin is not known. Results We identified a mutation of zebrafish dync1h1 in a forward genetic screen that caused a deficit of oligodendrocytes. Using in vivo imaging and gene expression analyses, we additionally found evidence that dync1h1 promotes axon ensheathment and myelin gene expression. Conclusions In addition to its well known roles in axon transport and neuronal migration, cytoplasmic dynein contributes to neural development by promoting myelination. PMID:25488883

  14. Nucleotide sequence and transcriptional analysis of the HindIII P region of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus.

    PubMed

    Carstens, E B; Lu, A

    1990-12-01

    DNA sequence analysis of the HindIII P region of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus confirmed the specific amplification of 1.4 kb of viral DNA from this region of the genome. The sequenced region included an open reading frame, translated in a counterclockwise direction, which would potentially encode a 74K protein. The amplified DNA was contained within this open reading frame, resulting in in-frame amplifications of a domain within the protein. Transcription studies revealed the presence of a ladder of viral RNA species corresponding to a 2.5 kb transcript carrying tandem repeats of about 1.4 kb. This indicated that the duplicated DNA was transcribed in the same orientation as the p10 gene. We predict that transcripts synthesized from the opposite DNA strand also consist of a ladder of related mRNAs which would be translated to produce a family of p74 proteins with multiple internal domains. PMID:2273394

  15. Baculovirus replication: characterization of DNA and proteins synthesized by a nuclear polyhedrosis virus of Lymantria dispar, the gypsy moth, in a homologous cell line

    SciTech Connect

    McClintock, J.T.

    1985-01-01

    A multiple-embedded nuclear polyhedrosis virus (NPV) of the gypsy moth, Lymantria dispar (LdMNPV), is used for biological control. However, LdMNPV has low natural virulence and a long infection cycle in relation to other NPVs. Therefore, the replicative cycle of LdMNPV was investigated using a homologous cell line, IPLB-LD-652Y. Based on analyses of virus growth curves LdMNPV nonoccluded virus and polyhedral inclusion bodies appeared approximately 20 and 50 hr postinfection (p.i.), respectively. LdMNPV polypeptides, identified by autoradiography of (/sup 35/S)-methionine labeled fractions in SDS-PAGE, were synthesized in sequential phases: (1) an early ..cap alpha.. phase of replication (4 polypeptides from 4 to 12 hr p.i.), (2) an intermediate ..beta.. phase (20 polypeptides from 12 to 24 hr p.i.), and a late ..gamma.. phase (4 polypeptides from 24 to 28 hr p.i.). In infected cells at least four polypeptides were post-translational cleaved and/or modified. Pulse-labeling with (/sup 3/H)-mannose, (/sup 3/H)-N-acetyl-glucosamine or (/sup 32/P)-monosodium phosphate revealed several viral polypeptides which were glycosylated and/or phosphorylated. DNA:DNA dot hybridization experiments suggested that LdMNPV DNA synthesis was initiated between 12 to 16 hr p.i., increasing significantly thereafter.

  16. The RING for gypsy moth control: Topical application of fragment of its nuclear polyhedrosis virus anti-apoptosis gene as insecticide.

    PubMed

    Oberemok, Volodymyr V; Laikova, Kateryna V; Zaitsev, Aleksei S; Gushchin, Vladimir A; Skorokhod, Oleksii A

    2016-07-01

    Numerous studies suggest a cellular origin for the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) anti-apoptosis genes IAPs, thus opening a possibility to use the fragments of these genes for modulation of host metabolism. We report here the strong insecticidal and metabolic effect of single-stranded antisense DNA fragment from RING (really interesting new gene) domain of gypsy moth LdMNPV IAP-3 gene: specifically, on reduction of biomass (by 35%) and survival of L. dispar caterpillars. The treatment with this DNA fragment leads to a significantly higher mortality rates of female insects (1.7 fold) accompanied with the signs of apoptosis. Additionally, we show increased expression of host IAP-1, caspase-4 and gelsolin genes in eggs laid by survived females treated with RING DNA fragment accompanied with calcium and magnesium imbalance, indicating that the strong stress reactions and metabolic effects are not confined to treated insects but likely led to apoptosis in eggs too. The proposed new approach for insect pest management, which can be considered as advancement of "microbial pesticides", is based on the application of the specific virus DNA, exploiting the knowledge about virus-pest interactions and putting it to the benefit of mankind. PMID:27265824

  17. Influence of cytochrome c on apoptosis induced by Anagrapha (Syngrapha) falcifera multiple nuclear polyhedrosis virus (AfMNPV) in insect Spodoptera litura cells.

    PubMed

    Liu, Lijun; Peng, Jianxin; Liu, Kaiyu; Yang, Hong; Li, Yi; Hong, Huazhu

    2007-09-01

    We investigated the influence of cytochrome c on apoptosis induced by Anagrapha (Syngrapha) falcifera multiple nuclear polyhedrosis virus (AfMNPV). Microscopic observation revealed that infection of SL-1 cells with AfMNPV resulted in apoptosis, displaying apoptotic bodies in fluorescent-stained nuclei of AfMNPV-infected SL-1cells. Western blot analysis demonstrated that AfMNPV-induced apoptosis in insect SL-1 cells was significantly inhibited by cyclosporin A which blocked a translocation of cytochrome c from the mitochondria to the cytosol. As determined by using AC-DEVD-AFC as substrate, the activity of caspase-3 in AfMNPV-induced cells was detected as early as 4h post infection, gradually increased with time extension, and reached a highest level after 16h of infection. However, activity of caspase-3 in apoptotic cells decreased in the presence of cyclosporin A (30microM), indicating that activation of caspase-3 in SfaMNPV-induced cells was dependent on the release of cytochrome c from the mitochondria. In addition, cyclosporin A could markedly inhibit mitochondrial transmembrane potential (DeltaPsim) disruption in undergoing apoptotic cells. These data indicate that cytochrome c plays a key role in AfMNPV-induced apoptosis in S. litura cells and may be required for caspase activation during the induction of apoptosis. PMID:17478109

  18. An integrated model for the nucleo-cytoplasmic transport of cytoplasmic poly(A)-binding proteins.

    PubMed

    Burgess, Hannah M; Gray, Nicola K

    2012-05-01

    Cytoplasmic poly(A)-binding proteins (PABPs) regulate mRNA stability and translation. Although predominantly localized in the cytoplasm, PABP proteins also cycle through the nucleus. Recent work has established that their steady-state localization can be altered by cellular stresses such as ultraviolet (UV) radiation, and infection by several viruses, resulting in nuclear accumulation of PABPs. Here, we present further evidence that their interaction with and release from mRNA and translation complexes are important in determining their sub-cellular distribution and propose an integrated model for regulated nucleo-cytoplasmic transport of PABPs. PMID:22896784

  19. Arrest of cytoplasmic streaming induces algal proliferation in green paramecia.

    PubMed

    Takahashi, Toshiyuki; Shirai, Yohji; Kosaka, Toshikazu; Hosoya, Hiroshi

    2007-01-01

    A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells. PMID:18159235

  20. Hybridization using cytoplasmic male sterility, cytoplasmic herbicide tolerance, and herbicide tolerance from nuclear genes

    SciTech Connect

    Beversdorf, W.D.; Erickson, L.R.; Grant, I.

    1987-04-14

    An improved process is described for producing a substantially homogeneous population of plants of a predetermined hybrid variety of crop which is capable of undergoing self-pollination and cross-pollination. The process comprises: growing in a first planting area a substantially random population of cytoplasmic male sterile plants which exhibit cytoplasmic herbicide tolerance to at least one Type A herbicide and exhibit tolerance to at least one Type B herbicide which is attributable solely to homozygous dominant nuclear genes and male fertile plants which are homozygous recessive maintainer plants for the cytoplasmic male sterile plants and which lack the cytoplasmic herbicide tolerance to at least one Type A herbicide and exhibit tolerance to at least one Type B herbicide attributable solely to the homozygous dominant nuclear genes.

  1. Nuclear and cytoplasmic actin in dinoflagellates.

    PubMed

    Soyer-Gobillard, M O; Ausseil, J; Géraud, M L

    1996-01-01

    Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the

  2. The primary structure of rat brain (cytoplasmic) dynein heavy chain, a cytoplasmic motor enzyme.

    PubMed Central

    Zhang, Z; Tanaka, Y; Nonaka, S; Aizawa, H; Kawasaki, H; Nakata, T; Hirokawa, N

    1993-01-01

    Overlapping cDNA clones encoding the heavy chain of rat brain cytoplasmic dynein have been isolated. The isolated cDNA clones contain an open reading frame of 13,932 bp encoding 4644 aa (M(r), 532,213). The deduced protein sequence of the heavy chain of rat brain dynein shows significant similarity to sea urchin flagellar beta-dynein (27.0% identical) and to Dictyostelium cytoplasmic dynein (53.5% identical) throughout the entire sequence. The heavy chain of rat brain (cytoplasmic) dynein contains four putative nucleotide-binding consensus sequences [GX4GK(T/S)] in the central one-third region that are highly similar to those of sea urchin and Dictyostelium dyneins. The N-terminal one-third of the heavy chain of rat brain (cytoplasmic) dynein shows high similarity (43.8% identical) to that of Dictyostelium cytoplasmic dynein but poor similarity (19.4% identical) to that of sea urchin flagellar dynein. These results suggested that the C-terminal two-thirds of the dynein molecule is conserved and plays an essential role in microtubule-dependent motility activity, whereas the N-terminal regions are different between cytoplasmic and flagellar dyneins. Images Fig. 1 PMID:7690137

  3. Mechanism of Cytoplasmic mRNA Translation

    PubMed Central

    2015-01-01

    Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

  4. Protein diffusion in mammalian cell cytoplasm.

    PubMed

    Kühn, Thomas; Ihalainen, Teemu O; Hyväluoma, Jari; Dross, Nicolas; Willman, Sami F; Langowski, Jörg; Vihinen-Ranta, Maija; Timonen, Jussi

    2011-01-01

    We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS. PMID:21886771

  5. Replication of simian virus 40 origin-containing DNA during infection with a recombinant Autographa californica multiple nuclear polyhedrosis virus expressing large T antigen.

    PubMed Central

    Martin, D W; Weber, P C

    1997-01-01

    Autographica californica multiple nuclear polyhedrosis virus (AcMNPV) has been shown to encode many of the enzymes involved in the replication of its own DNA. Although the AcMNPV genome contains multiple sets of reiterated sequences that are thought to function as origins of DNA replication, no initiator protein has yet been identified in the set of viral replication enzymes. In this study, the ability of a heterologous origin initiator system to promote DNA replication in AcMNPV-infected cells was examined. A recombinant AcMNPV that expressed the simian virus 40 (SV40) large T antigen was surprisingly found to induce the efficient replication of a transfected plasmid containing an SV40 origin. This replication was subsequently found to involve three essential components: (i) T antigen, since replication of SV40 origin-containing plasmids was not induced by wild-type AcMNPV which did not express this protein; (ii) an intact SV40 core origin, since deletion of specific functional motifs within the origin resulted in a loss of replicative abilities; and (iii) one or more AcMNPV-encoded proteins, since viral superinfection was required for plasmid amplification. Characterization of the replicated DNA revealed that it existed as a high-molecular-weight concatemer and underwent significant levels of homologous recombination between inverted repeat sequences. These properties were consistent with an AcMNPV-directed mode of DNA synthesis rather than that of SV40 and suggested that T antigen-SV40 origin complexes may be capable of initiating DNA replication reactions that can be completed by AcMNPV-encoded enzymes. PMID:8985377

  6. Connexin channel permeability to cytoplasmic molecules.

    PubMed

    Harris, Andrew L

    2007-01-01

    Connexin channels are known to be permeable to a variety of cytoplasmic molecules. The first observation of second messenger junctional permeability, made approximately 30 years ago, sparked broad interest in gap junction channels as mediators of intercellular molecular signaling. Since then, much has been learned about the diversity of connexin channels with regard to isoform diversity, tissue and developmental distribution, modes of channel regulation, assembly, expression, biochemical modification and permeability, all of which appear to be dynamically regulated. This information has expanded the potential roles of connexin channels in development, physiology and disease, and made their elucidation much more complex--30 years ago such an orchestra of junctional dynamics was unanticipated. Only recently, however, have investigators been able to directly address, in this more complex framework, the key issue: what specific biological molecules, second messengers and others, are able to permeate the various types of connexin channels, and how well? An important related issue, given the ever-growing list of connexin-related pathologies, is how these permeabilities are altered by disease-causing connexin mutations. Together, many studies show that a variety of cytoplasmic molecules can permeate the different types of connexin channels. A few studies reveal differences in permeation by different molecules through a particular type of connexin channel, and differences in permeation by a particular molecule through different types of connexin channels. This article describes and evaluates the various methods used to obtain these data, presents an annotated compilation of the results, and discusses the findings in the context of what can be inferred about mechanism of selectivity and potential relevance to signaling. The data strongly suggest that highly specific interactions take place between connexin pores and specific biological molecular permeants, and that those

  7. A model for the coordinated stepping of cytoplasmic dynein.

    PubMed

    Zhao, X Y; Sun, W; Zhang, J P; Tala; Guo, W S

    2014-10-31

    Cytoplasmic dynein play an important role in transporting various intracellular cargos by coupling their ATP hydrolysis cycle with their conformational changes. Recent experimental results showed that the cytoplasmic dynein had a highly variable stepping pattern including "hand-over-hand", "inchworm" and "nonalternating-inchworm". Here, we developed a model to describe the coordinated stepping patterns of cytoplasmic dynein, based on its working cycle, construction and the interaction between its leading head and tailing head. The kinetic model showed how change in the distance between the two heads influences the rate of cytoplasmic dynein under different stepping patterns. Numerical simulations of the distribution of step size and striding rate are in good quantitative agreement with experimental observations. Hence, our coordinated stepping model for cytoplasmic dynein successfully explained its diverse stepping patterns as a molecular motor. The cooperative mechanism carried out by the two heads of cytoplasmic dynein shed light on the strategies adopted by the cytoplasmic dynein in executing various functions. PMID:25301561

  8. PTEN functions by recruitment to cytoplasmic vesicles.

    PubMed

    Naguib, Adam; Bencze, Gyula; Cho, Hyejin; Zheng, Wu; Tocilj, Ante; Elkayam, Elad; Faehnle, Christopher R; Jaber, Nadia; Pratt, Christopher P; Chen, Muhan; Zong, Wei-Xing; Marks, Michael S; Joshua-Tor, Leemor; Pappin, Darryl J; Trotman, Lloyd C

    2015-04-16

    PTEN is proposed to function at the plasma membrane, where receptor tyrosine kinases are activated. However, the majority of PTEN is located throughout the cytoplasm. Here, we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via phosphatidylinositol 3-phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the non-catalytic C2 domain of PTEN specifically binds PI(3)P through the CBR3 loop. Mutations render this loop incapable of PI(3)P binding and abrogate PTEN-mediated inhibition of PI 3-kinase/AKT signaling. This loss of function is rescued by fusion of the loop mutant PTEN to FYVE, the canonical PI(3)P binding domain, demonstrating the functional importance of targeting PTEN to endosomal membranes. Beyond revealing an upstream activation mechanism of PTEN, our data introduce the concept of PI 3-kinase signal activation on the vast plasma membrane that is contrasted by PTEN-mediated signal termination on the small, discrete surfaces of internalized vesicles. PMID:25866245

  9. The epididymis, cytoplasmic droplets and male fertility

    PubMed Central

    Cooper, Trevor G

    2011-01-01

    The potential of spermatozoa to become motile during post-testicular maturation, and the relationship between the cytoplasmic droplet and fertilizing capacity are reviewed. Post-testicular maturation of spermatozoa involves the autonomous induction of motility, which can occur in vivo in testes with occluded excurrent ducts and in vitro in testicular explants, and artefactual changes in morphology that appear to occur in the testis in vitro. Both modifications may reflect time-dependent oxidation of disulphide bonds of head and tail proteins. Regulatory volume decrease (RVD), which counters sperm swelling at ejaculation, is discussed in relation to loss of cytoplasmic droplets and consequences for fertility. It is postulated that: (i) fertile males possess spermatozoa with sufficient osmolytes to drive RVD at ejaculation, permitting the droplet to round up and pinch off without membrane rupture; and (ii) infertile males possess spermatozoa with insufficient osmolytes so that RVD is inadequate, the droplet swells and the resulting flagellar angulation prevents droplet loss. Droplet retention at ejaculation is a harbinger of infertility caused by failure of the spermatozoon to negotiate the uterotubal junction or mucous and reach the egg. In this hypothesis, the epididymis regulates fertility indirectly by the extent of osmolyte provision to spermatozoa, which influences RVD and therefore droplet loss. Man is an exception, because ejaculated human spermatozoa retain their droplets. This may reflect their short midpiece, approximating head length, permitting a swollen droplet to extend along the entire midpiece; this not only obviates droplet migration and flagellar angulation but also hampers droplet loss. PMID:21076437

  10. Physical properties of cytoplasmic intermediate filaments.

    PubMed

    Block, Johanna; Schroeder, Viktor; Pawelzyk, Paul; Willenbacher, Norbert; Köster, Sarah

    2015-11-01

    Intermediate filaments (IFs) constitute a sophisticated filament system in the cytoplasm of eukaryotes. They form bundles and networks with adapted viscoelastic properties and are strongly interconnected with the other filament types, microfilaments and microtubules. IFs are cell type specific and apart from biochemical functions, they act as mechanical entities to provide stability and resilience to cells and tissues. We review the physical properties of these abundant structural proteins including both in vitro studies and cell experiments. IFs are hierarchical structures and their physical properties seem to a large part be encoded in the very specific architecture of the biopolymers. Thus, we begin our review by presenting the assembly mechanism, followed by the mechanical properties of individual filaments, network and structure formation due to electrostatic interactions, and eventually the mechanics of in vitro and cellular networks. This article is part of a Special Issue entitled: Mechanobiology. PMID:25975455

  11. Quantifying intermittent transport in cell cytoplasm

    NASA Astrophysics Data System (ADS)

    Lagache, Thibault; Holcman, David

    2008-03-01

    Active cellular transport is a fundamental mechanism for protein and vesicle delivery, cell cycle, and molecular degradation. Viruses can hijack the transport system and use it to reach the nucleus. Most transport processes consist of intermittent dynamics, where the motion of a particle, such as a virus, alternates between pure Brownian and directed movement along microtubules. In this Rapid Communication, we estimate the mean time for a particle to attach to a microtubule network. This computation leads to a coarse grained equation of the intermittent motion in radial and cylindrical geometries. Finally, by using the degradation activity inside the cytoplasm, we obtain refined asymptotic estimations for the probability and the mean time a virus reaches a small nuclear pore.

  12. Quantifying intermittent transport in cell cytoplasm.

    PubMed

    Lagache, Thibault; Holcman, David

    2008-03-01

    Active cellular transport is a fundamental mechanism for protein and vesicle delivery, cell cycle, and molecular degradation. Viruses can hijack the transport system and use it to reach the nucleus. Most transport processes consist of intermittent dynamics, where the motion of a particle, such as a virus, alternates between pure Brownian and directed movement along microtubules. In this Rapid Communication, we estimate the mean time for a particle to attach to a microtubule network. This computation leads to a coarse grained equation of the intermittent motion in radial and cylindrical geometries. Finally, by using the degradation activity inside the cytoplasm, we obtain refined asymptotic estimations for the probability and the mean time a virus reaches a small nuclear pore. PMID:18517320

  13. Non-ideal solution thermodynamics of cytoplasm.

    PubMed

    Ross-Rodriguez, Lisa U; Elliott, Janet A W; McGann, Locksley E

    2012-10-01

    Quantitative description of the non-ideal solution thermodynamics of the cytoplasm of a living mammalian cell is critically necessary in mathematical modeling of cryobiology and desiccation and other fields where the passive osmotic response of a cell plays a role. In the solution thermodynamics osmotic virial equation, the quadratic correction to the linear ideal, dilute solution theory is described by the second osmotic virial coefficient. Herein we report, for the first time, intracellular solution second osmotic virial coefficients for four cell types [TF-1 hematopoietic stem cells, human umbilical vein endothelial cells (HUVEC), porcine hepatocytes, and porcine chondrocytes] and further report second osmotic virial coefficients indistinguishable from zero (for the concentration range studied) for human hepatocytes and mouse oocytes. PMID:23840923

  14. Inborn errors of cytoplasmic triglyceride metabolism.

    PubMed

    Wu, Jiang Wei; Yang, Hao; Wang, Shu Pei; Soni, Krishnakant G; Brunel-Guitton, Catherine; Mitchell, Grant A

    2015-01-01

    Triglyceride (TG) synthesis, storage, and degradation together constitute cytoplasmic TG metabolism (CTGM). CTGM is mostly studied in adipocytes, where starting from glycerol-3-phosphate and fatty acyl (FA)-coenzyme A (CoA), TGs are synthesized then stored in cytoplasmic lipid droplets. TG hydrolysis proceeds sequentially, producing FAs and glycerol. Several reactions of CTGM can be catalyzed by more than one enzyme, creating great potential for complex tissue-specific physiology. In adipose tissue, CTGM provides FA as a systemic energy source during fasting and is related to obesity. Inborn errors and mouse models have demonstrated the importance of CTGM for non-adipose tissues, including skeletal muscle, myocardium and liver, because steatosis and dysfunction can occur. We discuss known inborn errors of CTGM, including deficiencies of: AGPAT2 (a form of generalized lipodystrophy), LPIN1 (childhood rhabdomyolysis), LPIN2 (an inflammatory condition, Majeed syndrome, described elsewhere in this issue), DGAT1 (protein loosing enteropathy), perilipin 1 (partial lipodystrophy), CGI-58 (gene ABHD5, neutral lipid storage disease (NLSD) with ichthyosis and "Jordan's anomaly" of vacuolated polymorphonuclear leukocytes), adipose triglyceride lipase (ATGL, gene PNPLA2, NLSD with myopathy, cardiomyopathy and Jordan's anomaly), hormone-sensitive lipase (HSL, gene LIPE, hypertriglyceridemia, and insulin resistance). Two inborn errors of glycerol metabolism are known: glycerol kinase (GK, causing pseudohypertriglyceridemia) and glycerol-3-phosphate dehydrogenase (GPD1, childhood hepatic steatosis). Mouse models often resemble human phenotypes but may diverge markedly. Inborn errors have been described for less than one-third of CTGM enzymes, and new phenotypes may yet be identified. PMID:25300978

  15. Internal Sense of Direction: Sensing and Signaling from Cytoplasmic Chemoreceptors

    PubMed Central

    Collins, Kieran D.; Lacal, Jesus

    2014-01-01

    SUMMARY Chemoreceptors sense environmental signals and drive chemotactic responses in Bacteria and Archaea. There are two main classes of chemoreceptors: integral inner membrane and soluble cytoplasmic proteins. The latter were identified more recently than integral membrane chemoreceptors and have been studied much less thoroughly. These cytoplasmic chemoreceptors are the subject of this review. Our analysis determined that 14% of bacterial and 43% of archaeal chemoreceptors are cytoplasmic, based on currently sequenced genomes. Cytoplasmic chemoreceptors appear to share the same key structural features as integral membrane chemoreceptors, including the formations of homodimers, trimers of dimers, and 12-nm hexagonal arrays within the cell. Cytoplasmic chemoreceptors exhibit varied subcellular locations, with some localizing to the poles and others appearing both cytoplasmic and polar. Some cytoplasmic chemoreceptors adopt more exotic locations, including the formations of exclusively internal clusters or moving dynamic clusters that coalesce at points of contact with other cells. Cytoplasmic chemoreceptors presumably sense signals within the cytoplasm and bear diverse signal input domains that are mostly N terminal to the domain that defines chemoreceptors, the so-called MA domain. Similar to the case for transmembrane receptors, our analysis suggests that the most common signal input domain is the PAS (Per-Arnt-Sim) domain, but a variety of other N-terminal domains exist. It is also common, however, for cytoplasmic chemoreceptors to have C-terminal domains that may function for signal input. The most common of these is the recently identified chemoreceptor zinc binding (CZB) domain, found in 8% of all cytoplasmic chemoreceptors. The widespread nature and diverse signal input domains suggest that these chemoreceptors can monitor a variety of cytoplasmically based signals, most of which remain to be determined. PMID:25428939

  16. Cytoplasmic Adenylation and Processing of Maternal RNA

    PubMed Central

    Slater, Isabel; Gillespie, David; Slater, D. W.

    1973-01-01

    Molecular hybridization between [3H]-poly(U) and unlabeled RNA prepared from sea urchin eggs and embryos has been used to contrast the subcellular localization as well as the size distribution of adenylylated maternal RNA preexisting in the unfertilized egg with that adenylylated as a function of fertilization. Evidence reported establishes that such preadenylylated genetic messages are predominantly located in the ovum's subribosomal fraction and that fertilization elicits a rapid reallocation of these latent transcripts into the zygote's ribosomal fraction. Examination of the size distribution of the adenylylated RNA further demonstrates that the unfertilized egg contains a substantial population of RNA transcripts of exceptionally high molecular weight that are used as primers for the 2-fold net synthesis of poly(A) that follows fertilization. The poly(A)-rich tracts are shown to be covalently bonded to RNA. Assessment of the poly(A) content of nuclear and cytoplasmic fractions suggests that the function of poly(A) is not confined to the transport of genetic messages from the nucleus. PMID:4510284

  17. Tropomyosin-Mediated Regulation of Cytoplasmic Myosins.

    PubMed

    Manstein, Dietmar J; Mulvihill, Daniel P

    2016-08-01

    The ability of the actin-based cytoskeleton to rapidly reorganize is critical for maintaining cell organization and viability. The plethora of activities in which actin polymers participate require different biophysical properties, which can vary significantly between the different events that often occur simultaneously at separate cellular locations. In order to modify the biophysical properties of an actin polymer for a particular function, the cell contains diverse actin-binding proteins that modulate the growth, regulation and molecular interactions of actin-based structures according to functional requirements. In metazoan and yeast cells, tropomyosin is a key regulator of actin-based structures. Cells have the capacity to produce multiple tropomyosin isoforms, each capable of specifically associating as copolymers with actin at distinct cellular locations to fine-tune the functional properties of discrete actin structures. Here, we present a unifying theory in which tropomyosin isoforms critically define the surface landscape of copolymers with cytoplasmic β- or γ-actin. Decoration of filamentous actin with different tropomyosin isoforms determines the identity and modulates the activity of the interacting myosin motor proteins. Conversely, changes in the nucleotide state of actin and posttranslational modifications affect the composition, morphology, subcellular localization and allosteric coupling of the associated actin-based superstructures. PMID:27060364

  18. Cytoplasmic dynein promotes HIV-1 uncoating.

    PubMed

    Pawlica, Paulina; Berthoux, Lionel

    2014-11-01

    Retroviral capsid (CA) cores undergo uncoating during their retrograde transport (toward the nucleus), and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC) using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable) CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating. PMID:25375884

  19. Molecular analysis of cytoplasmic male sterility

    SciTech Connect

    Hanson, M.R.

    1990-01-01

    The ultimate aims of the project are to understand the molecular mechanism of the disruption in pollen development which occurs in cytoplasmic male sterile plants and to understand the control of respiratory energy flow in the higher plant cell. A mitochondrial locus termed S-pcf segregates with sterility and with an alteration in respiration in Petunia. This cloned locus contains three genes, an abnormal fused gene termed pcf, a gene for a subunit of an NADH dehydrogenase complex, and a small ribosomal subunit protein. The pcf gene is comprised of partial sequences of ATPase subunit 9, cytochrome oxidase subunit II, and an unidentified reading frame. Components of the S-Pcf locus will be introduced into the nuclear of a fertile genotype under the control of appropriate regulatory signals, and polypeptide products of introduced genes will be directed to the mitochondrion with a transit peptide. By examining transgenic plants, we can determine what elements of the locus are critical for altered respiration or sterility. Such knowledge could explain how mitochondrial DNA affects pollen development in the large number of plant species which exhibit the agronomically important trait of male sterility. 10 refs., 3 figs.

  20. The synthesis of polyribonucleotides by cytoplasmic enzymes

    PubMed Central

    Wykes, J. R.; Smellie, R. M. S.

    1966-01-01

    1. The possibility that the cell cytoplasm contains enzymes catalysing the biosynthesis of RNA was investigated in fractions obtained by differential centrifugation of homogenates of Landschutz ascites-tumour cells. 2. The microsomal fraction was shown to be most active in incorporating UMP residues from [α-32P]UTP into polyribonucleotide material. 3. The same fraction also incorporated [3H]CTP, [3H]ATP and [3H]GTP separately and independently of the presence of complementary ribonucleoside 5′-triphosphates. 4. The reaction was promoted by the addition of RNA and showed an absolute requirement for Mg2+ ions. 5. Analysis of alkaline hydrolysates of the reaction products after the incorporation of [α-32P]UTP showed that most of the radioactivity was recovered in (2′,3′)-UMP residues irrespective of whether CTP, ATP and GTP were present in the reaction mixture. 6. Extraction of RNA from the reaction mixtures after the incorporation of [3H]ATP, [3H]GTP or [3H]CTP and analysis by sucrosedensity-gradient centrifugation showed no labelling of the ribosomal RNA. Radioactive material appeared between the 4s region and the meniscus of the sucrose gradient. In agreement with this observation, determinations of the chain length of the product showed that only short sequences of polynucleotides were synthesized. It is concluded that only homopolyribonucleotide synthesis is catalysed by the microsomal fractions and that there is little or no synthesis of RNA-like heteropolymers. PMID:5947148

  1. Evolution of Wolbachia cytoplasmic incompatibility types.

    PubMed

    Dobson, Stephen L

    2004-10-01

    The success of obligate endosymbiotic Wolbachia infections in insects is due in part to cytoplasmic incompatibility (CI), whereby Wolbachia bacteria manipulate host reproduction to promote their invasion and persistence within insect populations. The observed diversity of CI types raises the question of what the evolutionary pathways are by which a new CI type can evolve from an ancestral type. Prior evolutionary models assume that Wolbachia exists within a host individual as a clonal infection. While endosymbiotic theory predicts a general trend toward clonality, Wolbachia provides an exception in which there is selection to maintain diversity. Here, evolutionary trajectories are discussed that assume that a novel Wolbachia variant will co-exist with the original infection type within a host individual as a superinfection. Relative to prior models, this assumption relaxes requirements and allows additional pathways for the evolution of novel CI types. In addition to describing changes in the Wolbachia infection frequency associated with the hypothesized evolutionary events, the predicted impact of novel CI variants on the host population is also described. This impact, resulting from discordant evolutionary interests of symbiont and host, is discussed as a possible cause of Wolbachia loss from the host population or host population extinction. The latter is also discussed as the basis for an applied strategy for the suppression of insect pest populations. Model predictions are discussed relative to a recently published Wolbachia genome sequence and prior characterization of CI in naturally and artificially infected insects. PMID:15562682

  2. Substrate Specificity of Cytoplasmic N-Glycosyltransferase*

    PubMed Central

    Naegeli, Andreas; Michaud, Gaëlle; Schubert, Mario; Lin, Chia-Wei; Lizak, Christian; Darbre, Tamis; Reymond, Jean-Louis; Aebi, Markus

    2014-01-01

    N-Linked protein glycosylation is a very common post-translational modification that can be found in all kingdoms of life. The classical, highly conserved pathway entails the assembly of a lipid-linked oligosaccharide and its transfer to an asparagine residue in the sequon NX(S/T) of a secreted protein by the integral membrane protein oligosaccharyltransferase. A few species in the class of γ-proteobacteria encode a cytoplasmic N-glycosylation system mediated by a soluble N-glycosyltransferase (NGT). This enzyme uses nucleotide-activated sugars to modify asparagine residues with single monosaccharides. As these enzymes are not related to oligosaccharyltransferase, NGTs constitute a novel class of N-glycosylation catalyzing enzymes. To characterize the NGT-catalyzed reaction, we developed a sensitive and quantitative in vitro assay based on HPLC separation and quantification of fluorescently labeled substrate peptides. With this assay we were able to directly quantify glycopeptide formation by Actinobacillus pleuropneumoniae NGT and determine its substrate specificities: NGT turns over a number of different sugar donor substrates and allows for activation by both UDP and GDP. Quantitative analysis of peptide substrate turnover demonstrated a strikingly similar specificity as the classical, oligosaccharyltransferase-catalyzed N-glycosylation, with NX(S/T) sequons being the optimal NGT substrates. PMID:24962585

  3. Cytoplasmic mRNA turnover and ageing

    PubMed Central

    Borbolis, Fivos; Syntichaki, Popi

    2015-01-01

    Messenger RNA (mRNA) turnover that determines the lifetime of cytoplasmic mRNAs is a means to control gene expression under both normal and stress conditions, whereas its impact on ageing and age-related disorders has just become evident. Gene expression control is achieved at the level of the mRNA clearance as well as mRNA stability and accessibility to other molecules. All these processes are regulated by cis-acting motifs and trans-acting factors that determine the rates of translation and degradation of transcripts. Specific messenger RNA granules that harbor the mRNA decay machinery or various factors, involved in translational repression and transient storage of mRNAs, are also part of the mRNA fate regulation. Their assembly and function can be modulated to promote stress resistance to adverse conditions and over time affect the ageing process and the lifespan of the organism. Here, we provide insights into the complex relationships of ageing modulators and mRNA turnover mechanisms. PMID:26432921

  4. A physical perspective on cytoplasmic streaming

    PubMed Central

    Goldstein, Raymond E.; van de Meent, Jan-Willem

    2015-01-01

    Organisms show a remarkable range of sizes, yet the dimensions of a single cell rarely exceed 100 µm. While the physical and biological origins of this constraint remain poorly understood, exceptions to this rule give valuable insights. A well-known counterexample is the aquatic plant Chara, whose cells can exceed 10 cm in length and 1 mm in diameter. Two spiralling bands of molecular motors at the cell periphery drive the cellular fluid up and down at speeds up to 100 µm s−1, motion that has been hypothesized to mitigate the slowness of metabolite transport on these scales and to aid in homeostasis. This is the most organized instance of a broad class of continuous motions known as ‘cytoplasmic streaming’, found in a wide range of eukaryotic organisms—algae, plants, amoebae, nematodes and flies—often in unusually large cells. In this overview of the physics of this phenomenon, we examine the interplay between streaming, transport and cell size and discuss the possible role of self-organization phenomena in establishing the observed patterns of streaming. PMID:26464789

  5. Can paternal leakage maintain sexually antagonistic polymorphism in the cytoplasm?

    PubMed Central

    Kuijper, B; Lane, N; Pomiankowski, A

    2015-01-01

    A growing number of studies in multicellular organisms highlight low or moderate frequencies of paternal transmission of cytoplasmic organelles, including both mitochondria and chloroplasts. It is well established that strict maternal inheritance is selectively blind to cytoplasmic elements that are deleterious to males – ’mother's curse’. But it is not known how sensitive this conclusion is to slight levels of paternal cytoplasmic leakage. We assess the scope for polymorphism when individuals bear multiple cytoplasmic alleles in the presence of paternal leakage, bottlenecks and recurrent mutation. When fitness interactions among cytoplasmic elements within an individual are additive, we find that sexually antagonistic polymorphism is restricted to cases of strong selection on males. However, when fitness interactions among cytoplasmic elements are nonlinear, much more extensive polymorphism can be supported in the cytoplasm. In particular, mitochondrial mutants that have strong beneficial fitness effects in males and weak deleterious fitness effects in females when rare (i.e. ’reverse dominance’) are strongly favoured under paternal leakage. We discuss how such epistasis could arise through preferential segregation of mitochondria in sex-specific somatic tissues. Our analysis shows how paternal leakage can dampen the evolution of deleterious male effects associated with predominant maternal inheritance of cytoplasm, potentially explaining why ’mother's curse’ is less pervasive than predicted by earlier work. PMID:25653025

  6. Developing improved durum wheat germplasm by altering the cytoplasmic genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In eukaryotic organisms, nuclear and cytoplasmic genomes interact to drive cellular functions. These genomes have co-evolved to form specific nuclear-cytoplasmic interactions that are essential to the origin, success, and evolution of diploid and polyploid species. Hundreds of genetic diseases in h...

  7. Nucleotide sequence of Neurospora crassa cytoplasmic initiator tRNA.

    PubMed Central

    Gillum, A M; Hecker, L I; Silberklang, M; Schwartzbach, S D; RajBhandary, U L; Barnett, W E

    1977-01-01

    Initiator methionine tRNA from the cytoplasm of Neurospora crassa has been purified and sequenced. The sequence is: pAGCUGCAUm1GGCGCAGCGGAAGCGCM22GCY*GGGCUCAUt6AACCCGGAGm7GU (or D) - CACUCGAUCGm1AAACGAG*UUGCAGCUACCAOH. Similar to initiator tRNAs from the cytoplasm of other eukaryotes, this tRNA also contains the sequence -AUCG- instead of the usual -TphiCG (or A)- found in loop IV of other tRNAs. The sequence of the N. crassa cytoplasmic initiator tRNA is quite different from that of the corresponding mitochondrial initiator tRNA. Comparison of the sequence of N. crassa cytoplasmic initiator tRNA to those of yeast, wheat germ and vertebrate cytoplasmic initiator tRNA indicates that the sequences of the two fungal tRNAs are no more similar to each other than they are to those of other initiator tRNAs. Images PMID:146192

  8. Antineutrophil Cytoplasmic Antibodies Associated With Infective Endocarditis

    PubMed Central

    Langlois, Vincent; Lesourd, Anais; Girszyn, Nicolas; Ménard, Jean-Francois; Levesque, Hervé; Caron, Francois; Marie, Isabelle

    2016-01-01

    Abstract To determine the prevalence of antineutrophil cytoplasmic antibodies (ANCA) in patients with infective endocarditis (IE) in internal medicine; and to compare clinical and biochemical features and outcome between patients exhibiting IE with and without ANCA. Fifty consecutive patients with IE underwent ANCA testing. The medical records of these patients were reviewed. Of the 50 patients with IE, 12 exhibited ANCA (24%). ANCA-positive patients with IE exhibited: longer duration between the onset of first symptoms and IE diagnosis (P = 0.02); and more frequently: weight loss (P = 0.017) and renal impairment (P = 0.08), lower levels of C-reactive protein (P = 0.0009) and serum albumin (P = 0.0032), involvement of both aortic and mitral valves (P = 0.009), and longer hospital stay (P = 0.016). Under multivariate analysis, significant factors for ANCA-associated IE were: longer hospital stay (P = 0.004), lower level of serum albumin (P = 0.02), and multiple valve involvement (P = 0.04). Mortality rate was 25% in ANCA patients; death was because of IE complications in all these patients. Our study identifies a high prevalence of ANCA in unselected patients with IE in internal medicine (24%). Our findings further underscore that ANCA may be associated with a subacute form of IE leading to multiple valve involvement and more frequent renal impairment. Because death was due to IE complications in all patients, our data suggest that aggressive therapy may be required to improve such patients’ outcome. PMID:26817911

  9. Single cytoplasmic dynein molecule movements: characterization and comparison with kinesin.

    PubMed Central

    Wang, Z; Khan, S; Sheetz, M P

    1995-01-01

    Cytoplasmic dynein is a major microtubule motor for minus-end directed movements including retrograde axonal transport. To better understand the mechanism by which cytoplasmic dynein converts ATP energy into motility, we have analyzed the nanometer-level displacements of latex beads coated with low numbers of cytoplasmic dynein molecules. Cytoplasmic dynein-coated beads exhibited greater lateral movements among microtubule protofilaments (ave. 5.1 times/microns of displacement) compared with kinesin (ave. 0.9 times/micron). In addition, dynein moved rearward up to 100 nm over several hundred milliseconds, often in correlation with off-axis movements from one protofilament to another. We suggest that single molecules of cytoplasmic dynein move the beads because 1) there is a linear dependence of bead motility on dynein/bead ratio, 2) the binding of beads to microtubules studied by laser tweezers is best fit by a first-order Poisson, and 3) the run length histogram of dynein beads follows a first-order decay. At the cellular level, the greater disorder of cytoplasmic dynein movements may facilitate transport by decreasing the duration of collisions between kinesin and cytoplasmic dynein-powered vesicles. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 9 PMID:8580344

  10. Cytoplasmic streaming direction reverses in dividing Paramecium bursaria.

    PubMed

    Sikora, J; Wasik, A; Zajaczkowska, M

    1991-11-29

    Using the interference-contrast videomicroscopy the speed of cytoplasmic streaming was measured during the sequence of division stages in thigmotactically settled specimens of Paramecium bursaria. The speed of cytoplasmic flow gradually decreased during the first stages of binary fission and movement became indistinguishable at stage D(3). Almost at the same time cytoplasm started to move in the opposite direction, pushing or pulling the dividing micronucleus into the prospective posterior daughter cell and eventually stopped at stage D(5)-D(6). Further cell division events proceeded without detectable movement of cytoplasmic components. Cytoplasmic streaming in the normal interphase route was gradually restored in daughter cells about 30-40 min after cell separation. During the whole period of binary fission phagocytosis was arrested. Transportation and participation in the positioning of prospective micronuclei in daughter cells seems to be the main function of cytoplasmic streaming activity in cell division of Paramecium bursaria. The possible relationship between the stages of cytoskeleton transitions and the kinetics of cytoplasmic streaming associated with cell divison is discussed. PMID:23194845

  11. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    SciTech Connect

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  12. Mechanism of genotoxicity induced by targeted cytoplasmic irradiation

    PubMed Central

    Hong, M; Xu, A; Zhou, H; Wu, L; Randers-Pehrson, G; Santella, R M; Yu, Z; Hei, T K

    2010-01-01

    Background: Direct damage to DNA is generally accepted as the main initiator of mutation and cancer induced by environmental carcinogens or ionising radiation. However, there is accumulating evidence suggesting that extracellular/extranuclear targets may also have a key role in mediating the genotoxic effects of ionising radiation. As the possibility of a particle traversal through the cytoplasm is much higher than through the nuclei in environmental radiation exposure, the contribution to genotoxic damage from cytoplasmic irradiation should not be ignored in radiation risk estimation. Although targeted cytoplasmic irradiation has been shown to induce mutations in mammalian cells, the precise mechanism(s) underlying the mutagenic process is largely unknown. Methods: A microbeam that can target the cytoplasm of cells with high precision was used to study mechanisms involved in mediating the genotoxic effects in irradiated human–hamster hybrid (AL) cells. Results: Targeted cytoplasmic irradiation induces oxidative DNA damages and reactive nitrogen species (RNS) in AL cells. Lipid peroxidation, as determined by the induction of 4-hydroxynonenal was enhanced in irradiated cells, which could be suppressed by butylated hydroxyl toluene treatment. Moreover, cytoplasmic irradiation of AL cells increased expression of cyclooxygenase-2 (COX-2) and activation of extracellular signal-related kinase (ERK) pathway. Conclusion: We herein proposed a possible signalling pathway involving reactive oxygen/nitrogen species and COX-2 in the cytoplasmic irradiation-induced genotoxicity effect. PMID:20842121

  13. CYTOPLASMIC VACUOLIZATION RESPONSES TO CYTOPATHIC BOVINE VIRAL DIARRHOEA VIRUS

    PubMed Central

    Birk, Alexander V.; Dubovi, Edward J.; Cohen-Gould, Leona; Donis, Ruben; Szeto, Hazel. H.

    2008-01-01

    Bovine Viral Diarrhea Virus (BVDV) is a positive sense, single-stranded RNA virus which exhibits two biotypes in standard cell culture systems. The cytopathic strains of this virus (cpBVDV) induce dramatic cytoplasmic vacuolization in cell cultures, while infection with the non-cytopathic (NCP-BVDV) strains produces no overt changes in the host cells. Our results show that extensive cytoplasmic vacuolization is the earliest morphological change in response to cpBVDV infection in MDBK cells. Cells with extensive vacuolization showed no co-existing chromatin condensation, caspase activation, or loss of membrane integrity. In addition, the caspase inhibitor (zVAD-fmk), although improving cell viability of infected cells from 6.7±2.2% to 18.8±2.2%, did not prevent vacuolization. On the ultrastructural level, the virus-induced cytoplasmic vacuoles are single membrane structures containing organelles and cellular debris, which appear capable of fusing with other vacuoles and engulfing surrounding cytoplasmic materials. LysoTracker Red which marks lysosomes did not stain the virus-induced cytoplasmic vacuoles. In addition, this lysosomal dye could be observed in the cytoplasm of vacuolized cells, suggesting a lysosomal abnormality. Our data demonstrate that cpBVDV induced a novel cell death pathway in MDBK cells that is primarily associated with lysosomal dysfunction and the formation of phagocytic cytoplasmic vacuoles, and this mode of cell death is different from apoptosis and necrosis. PMID:18054406

  14. Functional insights from molecular modeling, docking, and dynamics study of a cypoviral RNA dependent RNA polymerase.

    PubMed

    Kundu, Anirban; Dutta, Anirudha; Biswas, Poulomi; Das, Amit Kumar; Ghosh, Ananta Kumar

    2015-09-01

    Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) contains 11 double stranded RNA genome segments and infects tasar silkworm A. mylitta. RNA-dependent RNA polymerase (RdRp) is reported as a key enzyme responsible for propagation of the virus in the host cell but its structure function relationship still remains elusive. Here a computational approach has been taken to compare sequence and secondary structure of AmCPV RdRp with other viral RdRps to identify consensus motifs. Then a reliable pairwise sequence alignment of AmCPV RdRp with its closest sequence structure homologue λ3 RdRp is done to predict three dimensional structure of AmCPV RdRp. After comparing with other structurally known viral RdRps, important sequence and/or structural features involved in substrate entry or binding, polymerase reaction and the product release events have been identified. A conserved RNA pentanucleotide (5'-AGAGC-3') at the 3'-end of virus genome is predicted as cis-acting signal for RNA synthesis and its docking and simulation study along with the model of AmCPV RdRp has allowed to predict mode of template binding by the viral polymerase. It is found that template RNA enters into the catalytic center through nine sequence-independent and two sequence-dependent interactions with the specific amino acid residues. However, number of sequence dependent interactions remains almost same during 10 nano-second simulation time while total number of interactions decreases. Further, docking of N(7)-methyl-GpppG (mRNA cap) on the model as well as prediction of RNA secondary structure has shown the template entry process in the active site. These findings have led to postulate the mechanism of RNA-dependent RNA polymerization process by AmCPV RdRp. To our knowledge, this is the first report to evaluate structure function relationship of a cypoviral RdRp. PMID:26264734

  15. CYTOPLASMIC MICROTUBULAR DYNAMIC AND CHROMATIN ORGANIZATION DURING MAMMALIAN OOCYTE MATURATION

    EPA Science Inventory

    Coordinated alterations in oocyte chromosome and microtubule disposition occur during oogenesis and oocyte maturation in the mammal. imely transitions in meiotic spindle and cytoplasmic microtubules, due to modifications in both the assembly competence of the tubulin pool and nuc...

  16. Stabilization and Degradation Mechanisms of Cytoplasmic Ataxin-1

    PubMed Central

    Kohiyama, Mayumi F.; Lagalwar, Sarita

    2015-01-01

    Aggregation-prone proteins in neurodegenerative disease disrupt cellular protein stabilization and degradation pathways. The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is caused by a coding polyglutamine expansion in the Ataxin-1 gene (ATXN1), which gives rise to the aggregation-prone mutant form of ATXN1 protein. Cerebellar Purkinje neurons, preferentially vulnerable in SCA1, produce ATXN1 protein in both cytoplasmic and nuclear compartments. Cytoplasmic stabilization of ATXN1 by phosphorylation and 14-3-3-mediated mechanisms ultimately drive translocation of the protein to the nucleus where aggregation may occur. However, experimental inhibition of phosphorylation and 14-3-3 binding results in rapid degradation of ATXN1, thus preventing nuclear translocation and cellular toxicity. The exact mechanism of cytoplasmic ATXN1 degradation is currently unknown; further investigation of degradation may provide future therapeutic targets. This review examines the present understanding of cytoplasmic ATXN1 stabilization and potential degradation mechanisms during normal and pathogenic states. PMID:27168726

  17. Nuclear Proteins Hijacked by Mammalian Cytoplasmic Plus Strand RNA Viruses

    PubMed Central

    Lloyd, Richard E.

    2015-01-01

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. PMID:25818028

  18. Population replacement in Culex fatigans by means of cytoplasmic incompatibility

    PubMed Central

    Curtis, C. F.

    1976-01-01

    Three experiments were carried out in field cages to test the principle of “transport” of a desirable gene or chromosome into a wild Culex fatigans population as a result of the sterility in cross-matings associated with cytoplasmic incompatibility. Cycling populations of Delhi origin were established in the cages and daily releases were made of the IS31B strain, which has Paris cytoplasm and carries a male-linked translocation. It was shown that, if sufficient releases were made to establish a majority of the Paris cytoplasmic type, complete replacement by this cytoplasmic type subsequently occurred. However, as a result of partial compatibility of males of the Delhi population with Paris females, “recombinant” males with Paris cytoplasm and no translocation were produced. In an experiment in which a continuous low rate of “immigration” of a strain of Delhi origin was simulated, a gradual increase of the Paris cytoplasm nontranslocated type occurred, and renewed IS31B releases were necessary after 5 months to restore the predominance of this type. The results are compared with computer predictions and discussed in relation to the transport of genes for filaria refractoriness or chromosome translocations into wild populations. PMID:1085660

  19. Growth signalobody selects functional intrabodies in the mammalian cytoplasm.

    PubMed

    Lee, Songhee; Kaku, Yoshihiro; Inoue, Satoshi; Nagamune, Teruyuki; Kawahara, Masahiro

    2016-03-01

    A versatile strategy to inhibit protein functions in the cytoplasmic environment is eagerly anticipated for drug discovery. In this study, we demonstrate a novel system to directly select functional intrabodies from a library in the mammalian cytoplasm. In this system, a target homo-oligomeric antigen is expressed together with a single-chain Fv (scFv) library that is linked to the cytoplasmic domain of a receptor tyrosine kinase (RTK) in the cytoplasm of murine interleukin-3 (IL-3)-dependent cells. As the tyrosine kinase is activated by dimerization, only scFv-RTK clones that can bind to the target antigen would be oligomerized and transduce a growth signal under the IL-3-deprived condition, which leads to selection of functional intrabodies. To demonstrate this system, we used rabies virus phosphoprotein (RV-P) that forms dimers in the cytoplasm as a target antigen. As a result, functional intrabodies were selected using our system from a naïve scFv library as well as from a pre-selected anti-RV-P library generated by phage display. This system may be applied for screening intrabodies that can prevent progression of various severe diseases. PMID:26647155

  20. Cytoplasmic pH influences cytoplasmic calcium in MC3T3-E1 osteoblast cells

    NASA Technical Reports Server (NTRS)

    Lin, H. S.; Hughes-Fulford, M.; Kumegawa, M.; Pitts, A. C.; Snowdowne, K. W.

    1993-01-01

    We found that the cytoplasmic concentration of calcium (Cai) of MC3T3-E1 osteoblasts was influenced by the type of pH buffer we used in the perfusing medium, suggesting that intracellular pH (pHi) might influence Cai. To study this effect, the Cai and pHi were monitored as we applied various experimental conditions known to change pHi. Exposure to NH4Cl caused a transient increase in both pHi and Cai without a change in extracellular pH (pHo). Decreasing pHo and pHi by lowering the bicarbonate concentration of the medium decreased Cai, and increasing pHi by the removal of 5% CO2 increased Cai. Clamping pHi to known values with 10 microM nigericin, a potassium proton ionophore, also influenced Cai: acid pHi lowered Cai, whereas alkaline pHi increased it. The rise in Cai appears to be very sensitive to the extracellular concentration of calcium, suggesting the existence of a pH-sensitive calcium influx mechanism. We conclude that physiologic changes in pH could modulate Cai by controlling the influx of calcium ions and could change the time course of the Cai transient associated with hormonal activation.

  1. Cytoplasmic dynein heavy chain: the servant of many masters

    PubMed Central

    Schiavo, Giampietro; Greensmith, Linda; Hafezparast, Majid; Fisher, Elizabeth M.C.

    2013-01-01

    Cytoplasmic dynein is the main retrograde motor in all eukaryotic cells. This complex comprises different subunits assembled on a cytoplasmic dynein heavy chain 1 (DYNC1H1) dimer. Cytoplasmic dynein is particularly important for neurons because it carries essential signals and organelles from distal sites to the cell body. In the past decade, several mouse models have helped to dissect the numerous functions of DYNC1H1. Additionally, several DYNC1H1 mutations have recently been found in human patients that give rise to a broad spectrum of developmental and midlife-onset disorders. Here, we discuss the effects of mutations of mouse and human DYNC1H1 and how these studies are giving us new insight into the many critical roles DYNC1H1 plays in the nervous system. PMID:24035135

  2. Biotoxicity assays for fruiting body lectins and other cytoplasmic proteins.

    PubMed

    Künzler, Markus; Bleuler-Martinez, Silvia; Butschi, Alex; Garbani, Mattia; Lüthy, Peter; Hengartner, Michael O; Aebi, Markus

    2010-01-01

    Recent studies suggest that a specific class of fungal lectins, commonly referred to as fruiting body lectins, play a role as effector molecules in the defense of fungi against predators and parasites. Hallmarks of these fungal lectins are their specific expression in reproductive structures, fruiting bodies, and/or sclerotia and their synthesis on free ribosomes in the cytoplasm. Fruiting body lectins are released upon damage of the fungal cell and bind to specific carbohydrate structures of predators and parasites, which leads to deterrence, inhibition of growth, and development or even killing of these organisms. Here, we describe assays to assess the toxicity of such lectins and other cytoplasmic proteins toward three different model organisms: the insect Aedes aegypti, the nematode Caenorhabditis elegans, and the amoeba Acanthamoeba castellanii. All three assays are based on heterologous expression of the examined proteins in the cytoplasm of Escherichia coli and feeding of these recombinant bacteria to omnivorous and bacterivorous organisms. PMID:20816208

  3. Nuclear export modulates the cytoplasmic Sir2 homologue Hst2

    PubMed Central

    Wilson, Jeanne M; Le, Viet Q; Zimmerman, Collin; Marmorstein, Ronen; Pillus, Lorraine

    2006-01-01

    Modulating transcription factors is crucial to executing sophisticated gene expression programs. The silent information regulator 2 (Sir2) family of NAD-dependent protein deacetylases influences transcription by targeting proteins such as histones, p53 and forkhead-box family transcription factors. Although apparently cytoplasmic, both mammalian SIRT2 and its yeast orthologue Hst2 have been implicated in transcriptional regulation. Here, we show that Hst2 moves between the nucleus and cytoplasm, but is largely cytoplasmic owing to efficient nuclear export. This nuclear exclusion is mediated by the exportin chromosomal region maintenance 1 (Crm1) and a putative leucine-rich nuclear export sequence in Hst2, which overlaps a unique autoregulatory helix. Disruption of Hst2 export shows that nuclear exclusion inhibits the activity of Hst2 as a transcriptional repressor. Our identification of putative nuclear export sequences in numerous vertebrate SIRT2 proteins shows that active nuclear export can be a conserved mechanism for regulating Sir2 homologues. PMID:17110954

  4. Cleavage of cytoplasm within the oligonucleate zoosporangia of allomyces macrogynus.

    PubMed

    Ji, Yunjeong; Song, Youngsun; Kim, Namhun; Youn, Hyunjoo; Kang, Minkook; Song, Yurim; Cho, Chungwon

    2014-01-01

    Allomyces macrogynus produces zoosporangia that discharge uninucleate zoospores after cleavage of multinucleate cytoplasm. Cleavage of cytoplasm within the oligonucleate zoosporangia of A. macrogynus was visualized by constructing three-dimensional models based on electron micrographs and confocal images. In oligonucleate zoosporangia, three adjacent nuclei can form three cleavage planes with a line of intersection of the planes. The position and boundary of the cleavage planes are thought to be determined by the relative positions of the nuclei. The establishment of three cleavage planes by cleavage membranes occurred sequentially, and the nuclear axis connecting the centers of two nuclei affected the development of cleavage membranes on each cleavage plane. In multinucleate zoosporangia, groups of three neighboring nuclei near the cell cortex may initiate the sequential establishment of cleavage planes and then may interact with the nuclei further from the cortex until the interactions of nuclei are propagated to the central region of the cytoplasm. PMID:24871589

  5. AMPK Signaling Results in Cytoplasmic Sequestration of p27

    PubMed Central

    Short, John D.; Houston, Kevin D.; Dere, Ruhee; Cai, Sheng-Li; Kim, Jinhee; Johnson, Charles L.; Broaddus, Russell R.; Shen, Jianjun; Miyamoto, Susie; Tamanoi, Fuyuhiko; Kwiatkowski, David; Mills, Gordon B.; Walker, Cheryl Lyn

    2009-01-01

    Tuberin, the Tsc2 gene product, integrates PI3K/MAPK (mitogenic) and LKB1/AMPK (energy) signaling pathways, and previous independent studies have shown that loss of tuberin is associated with elevated AMPK signaling and altered p27 function. In Tsc2-null tumors and tumor-derived cells from Eker rats, we observed elevated AMPK signaling and concordant cytoplasmic mislocalization of p27. Cytoplasmic localization of p27 in Tsc2-null cells was reversible pharmacologically using inhibitors of the LKB1/AMPK pathway, and localization of p27 to the cytoplasm could be induced directly by activating AMPK physiologically (glucose deprivation) or genetically (constitutively-active AMPK) in Tsc2-proficient cells. Furthermore, AMPK phosphorylated p27 in vitro on at least three sites including T170 near the NLS, and T170 was demonstrated to determine p27 localization in response to AMPK signaling. p27 functions in the nucleus to suppress Cdk2 activity, and has been reported to mediate an anti-apoptotic function when localized to the cytoplasm. We found that cells with elevated AMPK signaling and cytoplasmic p27 localization exhibited elevated Cdk2 activity, which could be suppressed by inhibiting AMPK signaling. In addition, cells with elevated AMPK signaling and cytoplasmic p27 localization were resistant to apoptosis, which could be overcome by inhibition of AMPK signaling and relocalization of p27 to the nucleus. These data demonstrate that AMPK signaling determines the subcellular localization of p27, and identifies loss of integration of pathways controlling energy balance, the cell cycle and apoptosis due to aberrant AMPK and p27 function as a feature of cells that have lost the Tsc2 tumor suppressor gene. PMID:18701472

  6. Transcription factor TFII-I conducts a cytoplasmic orchestra.

    PubMed

    Roy, Ananda L

    2006-11-21

    In response to extracellular ligands, surface receptor tyrosine kinases and G-protein-coupled receptors activate isoforms of phospholipase C (PLC) and initiate calcium signaling. PLC can activate expression of surface transient receptor potential channels (TRPC) such as TRPC3, which modulate calcium entry through the plasma membrane. A recent paper shows that competitive binding of cytoplasmic TFII-I, a transcription factor, to PLC-gamma results in inhibition of TRPC3-mediated agonist-induced Ca(2+) entry. These results establish a novel cytoplasmic function for TFII-I. PMID:17168565

  7. Nuclear repulsion enables division autonomy in a single cytoplasm

    PubMed Central

    Anderson, Cori A.; Eser, Umut; Korndorf, Therese; Borsuk, Mark E.; Skotheim, Jan M.; Gladfelter, Amy S.

    2014-01-01

    Summary Background Current models of cell cycle control, based on classic studies of fused cells, predict that nuclei in a shared cytoplasm respond to the same CDK activities to undergo synchronous cycling. However, synchrony is rarely observed in naturally occurring syncytia, such as the multinucleate fungus Ashbya gossypii. In this system, nuclei divide asynchronously raising the question of how nuclear timing differences are maintained despite sharing a common milieu. Results We observe that neighboring nuclei are highly variable in division cycle duration and neighbors repel one another to space apart and demarcate their own cytoplasmic territories. The size of these territories increases as a nucleus approaches mitosis and can influence cycling rates. This non-random nuclear spacing is regulated by microtubules and is required for nuclear asynchrony, as nuclei that transiently come in very close proximity will partially synchronize. Sister nuclei born of the same mitosis are generally not persistent neighbors over their lifetimes yet remarkably retain similar division cycle times. This indicates that nuclei carry a memory of their birth state that influences their division timing and supports that nuclei subdivide a common cytosol into functionally distinct yet mobile compartments. Conclusions These findings support that nuclei use cytoplasmic microtubules to establish “cells within cells.” Individual compartments appear to push against one another to compete for cytoplasmic territory and insulate the division cycle. This provides a mechanism by which syncytial nuclei can spatially organize cell cycle signaling and suggests size control can act in a system without physical boundaries. PMID:24094857

  8. Method for Confirming Cytoplasmic Delivery of RNA Aptamers

    PubMed Central

    Dickey, David D; Dassie, Justin P; Giangrande, Paloma H

    2016-01-01

    RNA aptamers are single-stranded RNA oligos that represent a powerful emerging technology with potential for treating numerous diseases. More recently, cell-targeted RNA aptamers have been developed for delivering RNA interference (RNAi) modulators (siRNAs and miRNAs) to specific diseased cells (e.g., cancer cells or HIV infected cells) in vitro and in vivo. However, despite initial promising reports, the broad application of this aptamer delivery technology awaits the development of methods that can verify and confirm delivery of aptamers to the cytoplasm of target cells where the RNAi machinery resides. We recently developed a functional assay (RIP assay) to confirm cellular uptake and subsequent cytoplasmic release of an RNA aptamer which binds to a cell surface receptor expressed on prostate cancer cells (PSMA). To assess cytoplasmic delivery, the aptamer was chemically conjugated to saporin, a ribosome inactivating protein toxin that is toxic to cells only when delivered to the cytoplasm (where it inhibits the ribosome) by a cell-targeting ligand (e.g., aptamer). Here, we describe the chemistry used to conjugate the aptamer to saporin and discuss a gel-based method to verify conjugation efficiency. We also detail an in vitro functional assay to confirm that the aptamer retains function following conjugation to saporin and describe a cellular assay to measure aptamer-mediated saporin-induced cytotoxicity. PMID:26472453

  9. Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

    PubMed Central

    Barros, Francisco; Domínguez, Pedro; de la Peña, Pilar

    2012-01-01

    The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered. PMID:22470342

  10. Optomechatronic System For Automated Intra Cytoplasmic Sperm Injection

    NASA Astrophysics Data System (ADS)

    Shulev, Assen; Tiankov, Tihomir; Ignatova, Detelina; Kostadinov, Kostadin; Roussev, Ilia; Trifonov, Dimitar; Penchev, Valentin

    2015-12-01

    This paper presents a complex optomechatronic system for In-Vitro Fertilization (IVF), offering almost complete automation of the Intra Cytoplasmic Sperm Injection (ICSI) procedure. The compound parts and sub-systems, as well as some of the computer vision algorithms, are described below. System capabilities for ICSI have been demonstrated on infertile oocyte cells.

  11. Structure of a Cytoplasmic 11-Subunit RNA Exosome Complex.

    PubMed

    Kowalinski, Eva; Kögel, Alexander; Ebert, Judith; Reichelt, Peter; Stegmann, Elisabeth; Habermann, Bianca; Conti, Elena

    2016-07-01

    The RNA exosome complex associates with nuclear and cytoplasmic cofactors to mediate the decay, surveillance, or processing of a wide variety of transcripts. In the cytoplasm, the conserved core of the exosome (Exo10) functions together with the conserved Ski complex. The interaction of S. cerevisiae Exo10 and Ski is not direct but requires a bridging cofactor, Ski7. Here, we report the 2.65 Å resolution structure of S. cerevisiae Exo10 bound to the interacting domain of Ski7. Extensive hydrophobic interactions rationalize the high affinity and stability of this complex, pointing to Ski7 as a constitutive component of the cytosolic exosome. Despite the absence of sequence homology, cytoplasmic Ski7 and nuclear Rrp6 bind Exo10 using similar surfaces and recognition motifs. Knowledge of the interacting residues in the yeast complexes allowed us to identify a splice variant of human HBS1-Like as a Ski7-like exosome-binding protein, revealing the evolutionary conservation of this cytoplasmic cofactor. PMID:27345150

  12. Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

    SciTech Connect

    Lloyd, Richard E.

    2015-05-15

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.

  13. Experimental Analysis of Cell Function Using Cytoplasmic Streaming

    ERIC Educational Resources Information Center

    Janssens, Peter; Waldhuber, Megan

    2012-01-01

    This laboratory exercise investigates the phenomenon of cytoplasmic streaming in the fresh water alga "Nitella". Students use the fungal toxin cytochalasin D, an inhibitor of actin polymerization, to investigate the mechanism of streaming. Students use simple statistical methods to analyze their data. Typical student data are provided. (Contains 3…

  14. Gravity-dependent polarity of cytoplasmic streaming in Nitellopsis

    NASA Technical Reports Server (NTRS)

    Wayne, R.; Staves, M. P.; Leopold, A. C.

    1990-01-01

    The internodal cells of the characean alga Nitellopsis obtusa were chosen to investigate the effect of gravity on cytoplasmic streaming. Horizontal cells exhibit streaming with equal velocities in both directions, whereas in vertically oriented cells, the downward-streaming cytoplasm flows ca. 10% faster than the upward-streaming cytoplasm. These results are independent of the orientation of the morphological top and bottom of the cell. We define the ratio of the velocity of the downward- to the upward-streaming cytoplasm as the polar ratio (PR). The normal polarity of a cell can be reversed (PR < 1) by treatment with neutral red (NR). The NR effect may be the result of membrane hyperpolarization, caused by the opening of K+ channels. The K+ channel blocker TEA Cl- inhibits the NR effect. External Ca2+ is required for normal graviresponsiveness. The [Ca2+] of the medium determines the polarity of cytoplasmic streaming. Less than 1 micromole Ca2+ resulted in a PR < 1 while greater than 1 micromole Ca2+ resulted in the normal gravity response. The voltage-dependent Ca(2+)-channel blocker, nifedipine, inhibited the gravity response in a reversible manner, while treatment with LaCl3 resulted in a PR < 1, indicating the presence of two types of Ca2+ channels. A new model for graviperception is presented in which the whole cell acts as the gravity sensor, and the plasma membrane acts as the gravireceptor. This is supported by ligation and UV irradiation experiments which indicate that the membranes at both ends of the cell are required for graviperception. The density of the external medium also affects the PR of Nitellopsis. Calculations are presented that indicate that the weight of the protoplasm may provide enough potential energy to open ion channels.

  15. An osmotic system within the cytoplasm of cells.

    PubMed

    OPIE, E L

    1948-05-01

    The cytoplasm of cells of the liver and of the kidney is in large part occupied by bodies which respond to the water content of these cells and are modified by dissolved substances in the surrounding fluid or by physical change such as freezing. These bodies, in part mitochondria but designated more broadly cytochondria, constitute an osmotic system within the cytoplasm of cells. When the specific gravity of liver or kidney tissue is used as an index of changes in the water content of tissue, swelling of cytochondria in general follows the intake of water but this relation may be modified by a variety of conditions. When liver that has been frozen and thawed is immersed in water, cytochondria become swollen though the containing cells diminish in size. Solutions of sodium and of potassium chloride isotonic with blood plasma cause delayed swelling of cells and cytochondria, greater with the potassium salt; solutions of calcium chloride of equal molar concentration cause immediate swelling of cells and cytochondria. The basophile material of the cytoplasm (ribonucleic acid and related substances) and the material that gives to mitochondria their characteristic stain are removed by immersion in water but their disappearance is retarded by isotonic solutions of sodium or of potassium chloride and further delayed by hypertonic solutions. When the intensity of staining reactions is diminished by the partial loss of basophile substance or of the distinctive mitochondrial material, these are found at the surfaces of the cytoplasmic bodies, held perhaps by adsorption. When water, isotonic solutions of sodium chloride, or Ringer's solution comes into contact with immersed liver, they remove basophile and mitochondrial material from a superficial zone and substances with similar staining reactions appear in the cytoplasm of cells at a deeper level. Osmotic changes in the cytoplasmic bodies may be reversible. When liver tissue which has been for a short time immersed in water

  16. Mechanodelivery of nanoparticles to the cytoplasm of living cells

    NASA Astrophysics Data System (ADS)

    Emerson, Nyssa T.; Hsia, Chih-Hao; Rafalska-Metcalf, Ilona U.; Yang, Haw

    2014-04-01

    Nanotechnology has opened up the opportunity to probe, sense, and manipulate the chemical environment of biological systems with an unprecedented level of spatiotemporal control. A major obstacle to the full realization of these novel technologies is the lack of a general, robust, and simple method for the delivery of arbitrary nanostructures to the cytoplasm of intact live cells. Here, we identify a new delivery modality, based on mechanical disruption of the plasma membrane, which efficiently mediates the delivery of nanoparticles to the cytoplasm of mammalian cells. We use two distinct execution modes, two adherent cell lines, and three sizes of semiconducting nanocrystals, or quantum dots, to demonstrate its applicability and effectiveness. As the underlying mechanism is purely physical, we anticipate that such ``mechanodelivery'' can be generalized to other modes of execution as well as to the cytoplasmic introduction of a structurally diverse array of functional nanomaterials.Nanotechnology has opened up the opportunity to probe, sense, and manipulate the chemical environment of biological systems with an unprecedented level of spatiotemporal control. A major obstacle to the full realization of these novel technologies is the lack of a general, robust, and simple method for the delivery of arbitrary nanostructures to the cytoplasm of intact live cells. Here, we identify a new delivery modality, based on mechanical disruption of the plasma membrane, which efficiently mediates the delivery of nanoparticles to the cytoplasm of mammalian cells. We use two distinct execution modes, two adherent cell lines, and three sizes of semiconducting nanocrystals, or quantum dots, to demonstrate its applicability and effectiveness. As the underlying mechanism is purely physical, we anticipate that such ``mechanodelivery'' can be generalized to other modes of execution as well as to the cytoplasmic introduction of a structurally diverse array of functional nanomaterials

  17. Nucleoporin Nup98 mediates galectin-3 nuclear-cytoplasmic trafficking

    SciTech Connect

    Funasaka, Tatsuyoshi; Balan, Vitaly; Raz, Avraham; Wong, Richard W.

    2013-04-26

    Highlights: •Nuclear pore protein Nup98 is a novel binding partner of galectin-3. •Nup98 transports galectin-3 into cytoplasm. •Nup98 depletion leads to galectin-3 nuclear transport and induces growth retardation. •Nup98 may involve in ß-catenin pathway through interaction with galectin-3. -- Abstract: Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus–cytoplasm transport of galectin-3, which is a ß-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the ß-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the ß-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to ß-catenin to inhibit transcriptional activity. Reduced expression of ß-catenin target genes is consistent with the Nup98 reduction and the galectin-3–nucleus translocation rate. Overall, the results show Nup98’s involvement in nuclear–cytoplasm translocation of galectin-3 and ß-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.

  18. Cytoplasmic Functions of the Tumor Suppressor p53

    PubMed Central

    Green, Douglas R.; Kroemer, Guido

    2010-01-01

    The principal tumor suppressor protein, p53, accumulates in cells in response to DNA damage, oncogene activation, and other stresses. It acts as a nuclear transcription factor that transactivates genes involved in apoptosis, cell cycle regulation, and numerous other processes. An emerging area of research unravels additional activities of p53 in the cytoplasm, where it triggers apoptosis and inhibits autophagy. These novel functions contribute to p53’s mission as a tumor suppressor. PMID:19407794

  19. Increased cytoplasm viscosity hampers aggregate polar segregation in Escherichia coli.

    PubMed

    Oliveira, Samuel M D; Neeli-Venkata, Ramakanth; Goncalves, Nadia S M; Santinha, João A; Martins, Leonardo; Tran, Huy; Mäkelä, Jarno; Gupta, Abhishekh; Barandas, Marilia; Häkkinen, Antti; Lloyd-Price, Jason; Fonseca, José M; Ribeiro, Andre S

    2016-02-01

    In Escherichia coli, under optimal conditions, protein aggregates associated with cellular aging are excluded from midcell by the nucleoid. We study the functionality of this process under sub-optimal temperatures from population and time lapse images of individual cells and aggregates and nucleoids within. We show that, as temperature decreases, aggregates become homogeneously distributed and uncorrelated with nucleoid size and location. We present evidence that this is due to increased cytoplasm viscosity, which weakens the anisotropy in aggregate displacements at the nucleoid borders that is responsible for their preference for polar localisation. Next, we show that in plasmolysed cells, which have increased cytoplasm viscosity, aggregates are also not preferentially located at the poles. Finally, we show that the inability of cells with increased viscosity to exclude aggregates from midcell results in enhanced aggregate concentration in between the nucleoids in cells close to dividing. This weakens the asymmetries in aggregate numbers between sister cells of subsequent generations required for rejuvenating cell lineages. We conclude that the process of exclusion of protein aggregates from midcell is not immune to stress conditions affecting the cytoplasm viscosity. The findings contribute to our understanding of E. coli's internal organisation and functioning, and its fragility to stressful conditions. PMID:26507787

  20. Cytoplasmic Dynamics Reveals Two Modes of Nucleoid-Dependent Mobility

    PubMed Central

    Stylianidou, Stella; Kuwada, Nathan J.; Wiggins, Paul A.

    2014-01-01

    It has been proposed that forces resulting from the physical exclusion of macromolecules from the bacterial nucleoid play a central role in organizing the bacterial cell, yet this proposal has not been quantitatively tested. To investigate this hypothesis, we mapped the generic motion of large protein complexes in the bacterial cytoplasm through quantitative analysis of thousands of complete cell-cycle trajectories of fluorescently tagged ectopic MS2-mRNA complexes. We find the motion of these complexes in the cytoplasm is strongly dependent on their spatial position along the long axis of the cell, and that their dynamics are consistent with a quantitative model that requires only nucleoid exclusion and membrane confinement. This analysis also reveals that the nucleoid increases the mobility of MS2-mRNA complexes, resulting in a fourfold increase in diffusion coefficients between regions of the lowest and highest nucleoid density. These data provide strong quantitative support for two modes of nucleoid action: the widely accepted mechanism of nucleoid exclusion in organizing the cell and a newly proposed mode, in which the nucleoid facilitates rapid motion throughout the cytoplasm. PMID:25468347

  1. Coilin Shuttles between the Nucleus and Cytoplasm In Xenopus Oocytes

    PubMed Central

    Bellini, Michel; Gall, Joseph G.

    1999-01-01

    Coiled bodies are discrete nuclear organelles often identified by the marker protein p80-coilin. Because coilin is not detected in the cytoplasm by immunofluorescence and Western blotting, it has been considered an exclusively nuclear protein. In the Xenopus germinal vesicle (GV), most coilin actually resides in the nucleoplasm, although it is highly concentrated in 50–100 coiled bodies. When affinity-purified anti-coilin antibodies were injected into the cytoplasm of oocytes, they could be detected in coiled bodies within 2–3 h. Coiled bodies were intensely labeled after 18 h, whereas other nuclear organelles remained negative. Because the nuclear envelope does not allow passive diffusion of immunoglobulins, this observation suggests that anti-coilin antibodies are imported into the nucleus as an antigen–antibody complex with coilin. Newly synthesized coilin is not required, because cycloheximide had no effect on nuclear import and subsequent targeting of the antibodies. Additional experiments with myc-tagged coilin and myc-tagged pyruvate kinase confirmed that coilin is a shuttling protein. The shuttling of Nopp140, NO38/B23, and nucleolin was easily demonstrated by the targeting of their respective antibodies to the nucleoli, whereas anti-SC35 did not enter the germinal vesicle. We suggest that coilin, perhaps in association with Nopp140, may function as part of a transport system between the cytoplasm and the coiled bodies. PMID:10512877

  2. The Interaction of Neurofilaments with the Microtubule Motor Cytoplasmic Dynein

    PubMed Central

    Wagner, Oliver I.; Ascaño, Jennifer; Tokito, Mariko; Leterrier, Jean-Francois; Janmey, Paul A.; Holzbaur, Erika L. F.

    2004-01-01

    Neurofilaments are synthesized in the cell body of neurons and transported outward along the axon via slow axonal transport. Direct observation of neurofilaments trafficking in live cells suggests that the slow outward rate of transport is due to the net effects of anterograde and retrograde microtubule motors pulling in opposition. Previous studies have suggested that cytoplasmic dynein is required for efficient neurofilament transport. In this study, we examine the interaction of neurofilaments with cytoplasmic dynein. We used fluid tapping mode atomic force microscopy to visualize single neurofilaments, microtubules, dynein/dynactin, and physical interactions between these neuronal components. AFM images suggest that neurofilaments act as cargo for dynein, associating with the base of the motor complex. Yeast two-hybrid and affinity chromatography assays confirm this hypothesis, indicating that neurofilament subunit M binds directly to dynein IC. This interaction is blocked by monoclonal antibodies directed either to NF-M or to dynein. Together these data suggest that a specific interaction between neurofilament subunit M and cytoplasmic dynein is involved in the saltatory bidirectional motility of neurofilaments undergoing axonal transport in the neuron. PMID:15342782

  3. Female breast carcinomas: nuclear and cytoplasmic proteins versus steroid receptors.

    PubMed

    Bryś, M; Romanowicz-Makowska, H; Nawrocka, A; Krajewska, W M

    2000-01-01

    Nuclear and cytoplasmic proteins of human female breast cancer were analysed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Oestrogen receptor and progesterone receptor expression was determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. The electropherograms were developed by silver nitrate staining and quantitative analysis was carried out by video densitometer using the software Gel-Pro Analyzer. Nuclear and cytoplasmic proteins of breast carcinomas and normal tissue differed both qualitatively and quantitatively. Nuclear polypeptides of 108, 53 and 48 kD as well as the 36 kD cytoplasmic polypeptide were specific for tumour samples, while the 51 kD nuclear polypeptide was detected only in normal tissue. Quantitative differences in band density were noted in the 32 kD nuclear polypeptide. This polypeptide was expressed in greatest concentration in infiltrating ductal carcinomas which also indicated the greatest oestrogen receptor gene expression. This relationship appeared to be statistically significant (p < 0.005). No correlations were evident between the 32 kD protein expression and the progesterone receptor gene expression in any of the tissue types examined, nor between the 32 kD protein and the patient's age or tumour grade. PMID:10756981

  4. Genetic studies on cytoplasmic male sterility in maize

    SciTech Connect

    Laughnan, J.R.

    1992-01-01

    Our research concerns the basic mechanisms of cytoplasmic male sterility (CMS) and fertility restoration in maize. The molecular determination of CMS is in the DNA of the mitochondria (mtDNA) but specific nuclear restorer-of-fertility (Rf) genes can overrule the male-sterile effect of the cytoplasm. Our approach to the study of the Rf genes is threefold. We are attempting to tag the cms-S Rf genes and the cms-T Rf2 gene with controlling elements (CEs). Since we have identified a number of spontaneous Rf genes for cms-S and have demonstrated that they are themselves transposable, we are also searching for cases in which an Rf gene is inserted into a wild-type gene. The other aspect of our research involves the nuclear control over the organization of the mitochondrial genome. We found that the changes in mtDNA organization upon cytoplasmic reversion to fertility were characteristic of the nuclear background in which the reversion event occurred. We have investigated whether these differences are a reflection of differences in the organization of the mtDNA genome before reversion.

  5. Cytoplasmic Control of Sense-Antisense mRNA Pairs.

    PubMed

    Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

    2015-09-22

    Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. PMID:26344770

  6. Cytoplasmic calcium buffers in intact human red cells.

    PubMed Central

    Tiffert, T; Lew, V L

    1997-01-01

    1. Precise knowledge of the cytoplasmic Ca2+ buffering behaviour in intact human red cells is essential for the characterization of their [Ca2+]i-dependent functions. This was investigated by using a refined method and experimental protocols which allowed continuity in the estimates of [Ca2+]i, from nanomolar to millimolar concentrations, in the presence and absence of external Ca2+ chelators. 2. The study was carried out in human red cells whose plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension. Cytoplasmic Ca2+ buffering was analysed from plots of total cell calcium content vs. ionized cytoplasmic Ca2+ concentration ([CaT]i vs. [Ca2+]i) obtained from measurements of the equilibrium distribution of 45Ca2+ at different external Ca2+ concentrations ([Ca2+]o), in conditions known to clamp cell volume and pH. The equilibrium distribution of 45Ca2+ was induced by the divalent cation ionophore A23187. 3. The results showed the following. (i) The known red cell Ca2+ buffer represented by alpha, with a large capacity and low Ca2+ affinity, was the main cytoplasmic Ca2+ binding agent. (ii) The value of alpha was remarkably constant; the means for each of four donors ranged from 0.33 to 0.35, with a combined value of all independent measurements of 0.34 +/- 0.01 (mean +/- S.E.M., n = 16). This contrasts with the variability previously reported. (iii) There was an additional Ca2+ buffering complex with a low capacity (approximately 80 micromol (340 g Hb)(-1)) and intermediate Ca2+ affinity (apparent dissociation constant, K(D,app) approximately 4-50 microM) whose possible identity is discussed. (iv) The cell content of putative Ca2+ buffers with submicromolar Ca2+ dissociation constants was below the detection limit of the methods used here (less than 2 micromol (340 g Hb)(-1)). 4. Vanadate (1 mM) inhibited the Vmax of the Ca2+ pump in inosine-fed cells by 99.7%. The cytoplasmic Ca2+ buffering behaviour

  7. DNA methylation affected by male sterile cytoplasm in rice (Oryza sativa L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Male sterile cytoplasm plays an important role in hybrid rice and cytoplasmic effects are sufficiently documented. However, no reports are available on DNA methylation affected by male sterile cytoplasm in hybrid rice. We used a methylation sensitive amplified polymorphism (MSAP) technique to charac...

  8. Pro-autophagic polyphenols reduce the acetylation of cytoplasmic proteins

    PubMed Central

    Pietrocola, Federico; Mariño, Guillermo; Lissa, Delphine; Vacchelli, Erika; Malik, Shoaib Ahmad; Niso-Santano, Mireia; Zamzami, Naoufal; Galluzzi, Lorenzo; Maiuri, Maria Chiara; Kroemer, Guido

    2012-01-01

    Resveratrol is a polyphenol contained in red wine that has been amply investigated for its beneficial effects on organismal metabolism, in particular in the context of the so-called “French paradox,” i.e., the relatively low incidence of coronary heart disease exhibited by a population with a high dietary intake of cholesterol and saturated fats. At least part of the beneficial effect of resveratrol on human health stems from its capacity to promote autophagy by activating the NAD-dependent deacetylase sirtuin 1. However, the concentration of resveratrol found in red wine is excessively low to account alone for the French paradox. Here, we investigated the possibility that other mono- and polyphenols contained in red wine might induce autophagy while affecting the acetylation levels of cellular proteins. Phenolic compounds found in red wine, including anthocyanins (oenin), stilbenoids (piceatannol), monophenols (caffeic acid, gallic acid) glucosides (delphinidin, kuronamin, peonidin) and flavonoids (catechin, epicatechin, quercetin, myricetin), were all capable of stimulating autophagy, although with dissimilar potencies. Importantly, a robust negative correlation could be established between autophagy induction and the acetylation levels of cytoplasmic proteins, as determined by a novel immunofluorescence staining protocol that allows for the exclusion of nuclear components from the analysis. Inhibition of sirtuin 1 by both pharmacological and genetic means abolished protein deacetylation and autophagy as stimulated by resveratrol, but not by piceatannol, indicating that these compounds act through distinct molecular pathways. In support of this notion, resveratrol and piceatannol synergized in inducing autophagy as well as in promoting cytoplasmic protein deacetylation. Our results highlight a cause-effect relationship between the deacetylation of cytoplasmic proteins and autophagy induction by red wine components. PMID:23070521

  9. NUCLEAR DNA AND CYTOPLASMIC DNA FROM TISSUES OF HIGHER PLANTS

    PubMed Central

    Hotta, Yasuo; Bassel, Alix; Stern, Herbert

    1965-01-01

    Young wheat roots were labeled with 32P-inorganic phosphate. Following the labeling period, roots were homogenized in a sucrose medium and fractionated into nuclei, cytoplasmic particles (including proplastids and mitochondria), and a soluble fraction containing most of the microsomes. DNA prepared from the particles had a higher buoyant density than that from the nuclei and showed a marked loss in total label if the roots were exposed to non-radioactive medium for 48 hours prior to fractionation of the cells. PMID:5885425

  10. Cytoplasmic and nuclear polyglutamine aggregates in SCA6 Purkinje cells.

    PubMed

    Ishikawa, K; Owada, K; Ishida, K; Fujigasaki, H; Shun Li, M; Tsunemi, T; Ohkoshi, N; Toru, S; Mizutani, T; Hayashi, M; Arai, N; Hasegawa, K; Kawanami, T; Kato, T; Makifuchi, T; Shoji, S; Tanabe, T; Mizusawa, H

    2001-06-26

    Aggregations of the alpha1A-calcium channel protein have been previously demonstrated in spinocerebellar ataxia type 6 (SCA6). Here the authors show that small aggregates, labeled by a monoclonal antibody 1C2 that preferentially detects expanded polyglutamine larger than that in SCA6 mutation, are present mainly in the cytoplasm but also in the nucleus of Purkinje cells. Although the length of expansion is small in SCA6, the current finding might indicate that SCA6 conforms to the pathogenic mechanism(s) in other polyglutamine diseases. PMID:11425948

  11. Cytoplasm affects grain weight and filled-grain ratio in indica rice

    PubMed Central

    2011-01-01

    Background Cytoplasmic effects on agronomic traits -involving cytoplasmic and nuclear genomes of either different species or different cultivars - are well documented in wheat but have seldom been demonstrated in rice (Oryza sativa L.). To detect cytoplasmic effects, we introgressed the nuclear genomes of three indica cultivars - Guichao 2, Jiangchengkugu, and Dianrui 449 - into the cytoplasms of six indica cultivars - Dijiaowujian, Shenglixian, Zhuzhan, Nantehao, Aizizhan, and Peta. These 18 nuclear substitution lines were evaluated during the winter season of 2005 in Sanya, Hainan, China, and during the summer season of 2006 in Kunming, Yunnan, China. The effects of 6 cytoplasm sources, 3 nucleus sources, 2 locations and their interactions were estimated for plant height, panicle length, panicle number per plant, spikelet number per panicle, grain weight, filled-grain ratio, and yield per plot. Results For five of the seven traits, analysis of variance showed that there were no significant cytoplasmic effects or interactions involving cytoplasmic effects. The effect of cytoplasm on 1000-grain weight was highly significant. Mean 1000-grain weight over the two locations in four of the six cytoplasms clustered close to the overall mean, whereas plants with Nantehao cytoplasm had a high, and those with Peta cytoplasm a low mean grain weight. There was a highly significant three-way interaction affecting filled-grain ratio. At Sanya, cytoplasms varied in very narrow ranges within nuclear backgrounds. Strong cytoplasmic effects were observed only at Kunming and in only two of the three nuclear backgrounds; in the Jianchenkugu nuclear background, there was no evidence of strong cytoplasmic effects at either location. In the Dianrui 449 and Guichao 2 nuclear background evaluated at Kunming, filled-grain ratios of the six cytoplasms showed striking rank shifts Conclusions We detected cytoplasmic variation for two agronomically important traits in indica rice. The

  12. Cytoplasmic Incompatibility and Bacterial Density in Nasonia Vitripennis

    PubMed Central

    Breeuwer, JAJ.; Werren, J. H.

    1993-01-01

    Cytoplasmically (maternally) inherited bacteria that cause reproductive incompatibility between strains are widespread among insects. In the parasitoid wasp Nasonia, incompatibility results in improper condensation and fragmentation of the paternal chromosomes in fertilized eggs. Some form of genome imprinting may be involved. Because of haplodiploidy, incompatibility results in conversion of (diploid) female eggs into (haploid) males. Experiments show that bacterial density is correlated with compatibility differences between male and female Nasonia. Males from strains with high bacterial numbers are incompatible with females from strains with lower numbers. Temporal changes in compatibility of females after tetracycline treatment are generally correlated with decreases in bacterial levels in eggs. However, complete loss of bacteria in mature eggs precedes conversion of eggs to the ``asymbiont'' compatibility type by 3-4 days. This result is consistent with a critical ``imprinting'' period during egg maturation, when cytoplasmic bacteria determine compatibility. Consequent inheritance of reduced bacterial numbers in F(1) progeny has different effects on compatibility type of subsequent male vs. female progeny. In some cases, partial incompatibility occurs which results in reduced offspring numbers, apparently due to incomplete paternal chromosome elimination resulting in aneuploidy. PMID:8244014

  13. Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

    PubMed

    Much, Christian; Auchynnikava, Tania; Pavlinic, Dinko; Buness, Andreas; Rappsilber, Juri; Benes, Vladimir; Allshire, Robin; O'Carroll, Dónal

    2016-06-01

    Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse. PMID:27254021

  14. CryoEM structure of yeast cytoplasmic exosome complex.

    PubMed

    Liu, Jun-Jie; Niu, Chu-Ya; Wu, Yao; Tan, Dan; Wang, Yang; Ye, Ming-Da; Liu, Yang; Zhao, Wenwei; Zhou, Ke; Liu, Quan-Sheng; Dai, Junbiao; Yang, Xuerui; Dong, Meng-Qiu; Huang, Niu; Wang, Hong-Wei

    2016-07-01

    The eukaryotic multi-subunit RNA exosome complex plays crucial roles in 3'-to-5' RNA processing and decay. Rrp6 and Ski7 are the major cofactors for the nuclear and cytoplasmic exosomes, respectively. In the cytoplasm, Ski7 helps the exosome to target mRNAs for degradation and turnover via a through-core pathway. However, the interaction between Ski7 and the exosome complex has remained unclear. The transaction of RNA substrates within the exosome is also elusive. In this work, we used single-particle cryo-electron microscopy to solve the structures of the Ski7-exosome complex in RNA-free and RNA-bound forms at resolutions of 4.2 Å and 5.8 Å, respectively. These structures reveal that the N-terminal domain of Ski7 adopts a structural arrangement and interacts with the exosome in a similar fashion to the C-terminal domain of nuclear Rrp6. Further structural analysis of exosomes with RNA substrates harboring 3' overhangs of different length suggests a switch mechanism of RNA-induced exosome activation in the through-core pathway of RNA processing. PMID:27174052

  15. A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis.

    PubMed

    Boutte, Cara C; Baer, Christina E; Papavinasasundaram, Kadamba; Liu, Weiru; Chase, Michael R; Meniche, Xavier; Fortune, Sarah M; Sassetti, Christopher M; Ioerger, Thomas R; Rubin, Eric J

    2016-01-01

    Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. A phylogenetic analysis of cwlM implies that localization in the cytoplasm drove the evolution of this factor. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance. PMID:27304077

  16. Sodium channels in the cytoplasm of Schwann cells.

    PubMed Central

    Ritchie, J M; Black, J A; Waxman, S G; Angelides, K J

    1990-01-01

    Immunoblotting, ultrastructural immunocytochemistry, and tritiated saxitoxin ([3H]STX) binding experiments were used to study sodium channel localization in Schwann cells. Polyclonal antibody 7493, which is directed against purified sodium channels from rat brain, specifically recognizes a 260-kDa protein corresponding to the alpha subunit of the sodium channel in immunoblots of crude glycoproteins from rat sciatic nerve. Electron microscopic localization of sodium channel immunoreactivity within adult rat sciatic nerves reveals heavy staining of the axon membrane at the node of Ranvier, in contrast to the internodal axon membrane, which does not stain. Schwann cells including perinodal processes also exhibit antibody 7493 immunoreactivity, localized within both the cytoplasm and the plasmalemma of the Schwann cell. To examine further the possibility that sodium channels are localized within Schwann cell cytoplasm, [3H]STX binding was studied in cultured rabbit Schwann cells, both intact and after homogenization. Saturable binding of STX was significantly higher in homogenized Schwann cells (410 +/- 37 fmol/mg of protein) than in intact Schwann cells (214 +/- 21 fmol/mg of protein). Moreover, the equilibrium dissociation constant was higher for homogenized preparations (1.77 +/- 0.37 nM) than for intact Schwann cells (1.06 +/- 0.29 nM). These data suggest the presence of an intracellular pool of sodium channels or channel precursors in Schwann cells. Images PMID:2174558

  17. A Balance between Nuclear and Cytoplasmic Volumes Controls Spindle Length

    PubMed Central

    Novakova, Lucia; Kovacovicova, Kristina; Dang-Nguyen, Thanh Quang; Sodek, Martin; Skultety, Michal; Anger, Martin

    2016-01-01

    Proper assembly of the spindle apparatus is crucially important for faithful chromosome segregation during anaphase. Thanks to the effort over the last decades, we have very detailed information about many events leading to spindle assembly and chromosome segregation, however we still do not understand certain aspects, including, for example, spindle length control. When tight regulation of spindle size is lost, chromosome segregation errors emerge. Currently, there are several hypotheses trying to explain the molecular mechanism of spindle length control. The number of kinetochores, activity of molecular rulers, intracellular gradients, cell size, limiting spindle components, and the balance of the spindle forces seem to contribute to spindle size regulation, however some of these mechanisms are likely specific to a particular cell type. In search for a general regulatory mechanism, in our study we focused on the role of cell size and nuclear to cytoplasmic ratio in this process. To this end, we used relatively large cells isolated from 2-cell mouse embryos. Our results showed that the spindle size upper limit is not reached in these cells and suggest that accurate control of spindle length requires balanced ratio between nuclear and cytoplasmic volumes. PMID:26886125

  18. Cytoplasm dynamics and cell motion: two-phase flow models.

    PubMed

    Alt, W; Dembo, M

    1999-03-01

    The motion of amoeboid cells is characterized by cytoplasmic streaming and by membrane protrusions and retractions which occur even in the absence of interactions with a substratum. Cell translocation requires, in addition, a transmission mechanism wherein the power produced by the cytoplasmic engine is applied to the substratum in a highly controlled fashion through specific adhesion proteins. Here we present a simple mechano-chemical model that tries to capture the physical essence of these complex biomolecular processes. Our model is based on the continuum equations for a viscous and reactive two-phase fluid model with moving boundaries, and on force balance equations that average the stochastic interactions between actin polymers and membrane proteins. In this paper we present a new derivation and analysis of these equations based on minimization of a power functional. This derivation also leads to a clear formulation and classification of the kinds of boundary conditions that should be specified at free surfaces and at the sites of interaction of the cell and the substratum. Numerical simulations of a one-dimensional lamella reveal that even this extremely simplified model is capable of producing several typical features of cell motility. These include periodic 'ruffle' formation, protrusion-retraction cycles, centripetal flow and cell-substratum traction forces. PMID:10204394

  19. Cytoplasmic translocation of the retinoblastoma protein disrupts sarcomeric organization.

    PubMed

    Araki, Keigo; Kawauchi, Keiko; Hirata, Hiroaki; Yamamoto, Mie; Taya, Yoichi

    2013-01-01

    Skeletal muscle degeneration is a complication arising from a variety of chronic diseases including advanced cancer. Pro-inflammatory cytokine TNF-α plays a pivotal role in mediating cancer-related skeletal muscle degeneration. Here, we show a novel function for retinoblastoma protein (Rb), where Rb causes sarcomeric disorganization. In human skeletal muscle myotubes (HSMMs), up-regulation of cyclin-dependent kinase 4 (CDK4) and concomitant phosphorylation of Rb was induced by TNF-α treatment, resulting in the translocation of phosphorylated Rb to the cytoplasm. Moreover, induced expression of the nuclear exporting signal (NES)-fused form of Rb caused disruption of sarcomeric organization. We identified mammalian diaphanous-related formin 1 (mDia1), a potent actin nucleation factor, as a binding partner of cytoplasmic Rb and found that mDia1 helps maintain the structural integrity of the sarcomere. These results reveal a novel non-nuclear function for Rb and suggest a potential mechanism of TNF-α-induced disruption of sarcomeric organization. DOI: http://dx.doi.org/10.7554/eLife.01228.001. PMID:24302570

  20. Cytoplasmic protein methylation is essential for neural crest migration

    PubMed Central

    Vermillion, Katie L.; Lidberg, Kevin A.

    2014-01-01

    As they initiate migration in vertebrate embryos, neural crest cells are enriched for methylation cycle enzymes, including S-adenosylhomocysteine hydrolase (SAHH), the only known enzyme to hydrolyze the feedback inhibitor of trans-methylation reactions. The importance of methylation in neural crest migration is unknown. Here, we show that SAHH is required for emigration of polarized neural crest cells, indicating that methylation is essential for neural crest migration. Although nuclear histone methylation regulates neural crest gene expression, SAHH and lysine-methylated proteins are abundant in the cytoplasm of migratory neural crest cells. Proteomic profiling of cytoplasmic, lysine-methylated proteins from migratory neural crest cells identified 182 proteins, several of which are cytoskeleton related. A methylation-resistant form of one of these proteins, the actin-binding protein elongation factor 1 alpha 1 (EF1α1), blocks neural crest migration. Altogether, these data reveal a novel and essential role for post-translational nonhistone protein methylation during neural crest migration and define a previously unknown requirement for EF1α1 methylation in migration. PMID:24379414

  1. Ulcerative colitis and antineutrophil cytoplasmic antibodies in Hong Kong Chinese.

    PubMed

    Sung, J Y; Chan, K L; Hsu, R; Liew, C T; Lawton, J W

    1993-06-01

    Inflammatory bowel diseases are known to be rare among the Chinese. The diagnosis of ulcerative colitis has been difficult in some of the Asian countries where infective colitis is more prevalent. Twenty-three Hong Kong Chinese patients diagnosed to have ulcerative colitis were reviewed. The symptoms were relatively mild and extraintestinal manifestation had been rare. Patients responded well to steroid therapy and sulfasalazine. Three patients in this series were found to have cyst and/or trophozoites of Entamoeba histolytica in stool. In this series, 19 patients were tested for antineutrophil cytoplasmic antibody (ANCA). Fourteen patients (73.5%) were positive, of which six (31.5%) showed a perinuclear staining pattern and eight (42%) demonstrated a cytoplasmic pattern. Five patients (26.5%) were negative for any ANCA, and none was positive for both. Sera of these patients were also tested for anti-alpha granules, anti-myeloperoxidase, and anti-lactoferrin activities. None was positive. Control sera collected from 16 patients with irritable bowel syndrome were all negative for the tests. In conclusion, testing of ANCAs may help in making the diagnosis of idiopathic inflammatory bowel disease in difficult situations. PMID:8503382

  2. Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity

    PubMed Central

    2015-01-01

    Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure–activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes. PMID:26555042

  3. A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis

    PubMed Central

    Boutte, Cara C; Baer, Christina E; Papavinasasundaram, Kadamba; Liu, Weiru; Chase, Michael R; Meniche, Xavier; Fortune, Sarah M; Sassetti, Christopher M; Ioerger, Thomas R; Rubin, Eric J

    2016-01-01

    Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. A phylogenetic analysis of cwlM implies that localization in the cytoplasm drove the evolution of this factor. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance. DOI: http://dx.doi.org/10.7554/eLife.14590.001 PMID:27304077

  4. Human cytoplasmic actin proteins are encoded by a multigene family

    SciTech Connect

    Engel, J.; Gunning, P.; Kedes, L.

    1982-06-01

    The authors characterized nine human actin genes that they isolated from a library of cloned human DNA. Measurements of the thermal stability of hybrids formed between each cloned actin gene and ..cap alpha..-, ..beta..-, and ..gamma..-actin mRNA demonstrated that only one of the clones is most homologous to sarcomeric actin mRNA, whereas the remaining eight clones are most homologous to cytoplasmic actin mRNA. By the following criteria they show that these nine clones represent nine different actin gene loci rather than different alleles or different parts of a single gene: (i) the restriction enzyme maps of the coding regions are dissimilar; (ii) each clone contains sufficient coding region to encode all or most of an entire actin gene; and (iii) each clone contains sequences homologous to both the 5' and 3' ends of the coding region of a cloned chicken ..beta..-actin cDNA. They conclude, therefore, that the human cytoplasmic actin proteins are encoded by a multigene family.

  5. Sodium channels in the cytoplasm of Schwann cells

    SciTech Connect

    Ritchie, J.M. ); Black, J.A.; Waxman, S.G. Veterans Affairs Medical Center, West Haven, CT ); Angelides, K.J. )

    1990-12-01

    Immunoblotting, ultrastructural immunocytochemistry, and tritiated saxitoxin (({sup 3}H)STX) binding experiments were used to study sodium channel localization in Schwann cells. Polyclonal antibody 7493, which is directed against purifed sodium channels from rat brain, specifically recognized a 260-kDa protein corresponding to the {alpha} subunit of the sodium channel in immunoblots of crude glycoproteins from rat sciatic nerve. Electron microscopic localization of sodium channel immunoreactivity within adult rat sciatic nerves reveals heavy staining of the axon membrane at the node of Ranvier, in contrast to the internodal axon membrane, which does not stain. Schwann cells including perinodal processes also exhibit antibody 7493 immunoreactivity, localized within both the cytoplasm and the plasmalemma of the Schwann cell. To examine further the possibility that sodium channels are localized within Schwann cell cytoplasm, ({sup 3}H)STX binding was studied in cultured rabbit Schwann cells, both intact and after homogenization. Saturable binding of STX was singificantly higher in homogenized Schwann cells than in intact Schwann cells. Moreover, the equilibrium dissociation constant was higher for homogenized preparations (1.77 {plus minus} 0.37 nM) than for intact Schwann cells (1.06 {plus minus} 0.29 nM). These data suggest the presence of an intracellular pool of sodium channels or channel presursors in Schwann cells.

  6. Antineutrophil cytoplasmic antibodies (ANCA) testing: detection methods and clinical application.

    PubMed

    Sinico, Renato Alberto; Radice, Antonella

    2014-01-01

    Antineutrophil cytoplasmic antibodies (ANCA) are considered the diagnostic biomarker of some necrotising vasculitis such as granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and, to a lesser extent, eosinophilic granulomatosis with polyangiitis (EGPA). According to the current recommendations, combining indirect immunofluorescence and proteinase 3 (PR3) and myeloperoxidase (MPO) antigen specific immunometric assays, in the proper clinical setting, assures the best diagnostic specificity. When such conditions are satisfied, ANCA are detected in up to 90% of patients with active generalised GPA and MPA and in about 40% of patients with EGPA. Cytoplasmic ANCA (C-ANCA) with specificity for PR3 are usually found in patients with GPA whereas perinuclear ANCA (P-ANCA) in patients with MPA and EGPA. However, ANCA antigen specificity is more closely associated with disease phenotype and prognosis than clinical diagnosis. The clinical value of serial ANCA testing in monitoring disease activity is still debated. Recently, new promising developments in methodology and techniques (computer-based image analysis of immunofluorescence patterns, novel generation of PR3-/MPO-ANCA immunometric assays and multiplex technology) have been proposed but studies comparing the performances of the different assays are scarce. PMID:24854381

  7. Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein

    PubMed Central

    Much, Christian; Pavlinic, Dinko; Buness, Andreas; Rappsilber, Juri; Benes, Vladimir; Allshire, Robin; O’Carroll, Dónal

    2016-01-01

    Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse. PMID:27254021

  8. A meta-analysis of the strength and nature of cytoplasmic genetic effects.

    PubMed

    Dobler, R; Rogell, B; Budar, F; Dowling, D K

    2014-10-01

    Genetic variation in cytoplasmic genomes (i.e. the mitochondrial genome in animals, and the combined mitochondrial and chloroplast genomes in plants) was traditionally assumed to accumulate under a neutral equilibrium model. This view has, however, come under increasing challenge from studies that have experimentally linked cytoplasmic genetic effects to the expression of life history phenotypes. Such results suggest that genetic variance located within the cytoplasm might be of evolutionary importance and potentially involved in shaping population evolutionary trajectories. As a step towards assessing this assertion, here we conduct a formal meta-analytic review to quantitatively assess the extent to which cytoplasmic genetic effects contribute to phenotypic expression across animal and plant kingdoms. We report that cytoplasmic effect sizes are generally moderate in size and associated with variation across a range of factors. Specifically, cytoplasmic effects on morphological traits are generally larger than those on life history or metabolic traits. Cytoplasmic effect sizes estimated at the between-species scale (via interspecies mix-and-matching of cytoplasmic and nuclear genomes) are larger than those at the within-species scale. Furthermore, cytoplasmic effects tied to epistatic interactions with the nuclear genome tend to be stronger than additive cytoplasmic effects, at least when restricting the data set to gonochorous animal species. Our results thus confirm that cytoplasmic genetic variation is commonly tied to phenotypic expression across plants and animals, implicate the cytoplasmic-nuclear interaction as a key unit on which natural selection acts and generally suggest that the genetic variation that lies within the cytoplasm is likely to be entwined in adaptive evolutionary processes. PMID:25196503

  9. The kinase domain of mitochondrial PINK1 faces the cytoplasm

    PubMed Central

    Zhou, Chun; Huang, Yong; Shao, Yufang; May, Jessica; Prou, Delphine; Perier, Celine; Dauer, William; Schon, Eric A.; Przedborski, Serge

    2008-01-01

    Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of autosomal recessive familial Parkinson's disease (PD). Efforts in deducing the PINK1 signaling pathway have been hindered by controversy around its subcellular and submitochondrial localization and the authenticity of its reported substrates. We show here that this mitochondrial protein exhibits a topology in which the kinase domain faces the cytoplasm and the N-terminal tail is inside the mitochondria. Although deletion of the transmembrane domain disrupts this topology, common PD-linked PINK1 mutations do not. These results are critical in rectifying the location and orientation of PINK1 in mitochondria, and they should help decipher its normal physiological function and potential pathogenic role in PD. PMID:18687899

  10. The Genes for Cytoplasmic Ribosomal Ribonucleic Acid in Higher Plants

    PubMed Central

    Scott, N. Steele; Ingle, J.

    1973-01-01

    The genes for cytoplasmic ribosomal RNA are partially resolved from the bulk of the DNA by CsCl equilibrium centrifugation. Although in some plants the buoyant density of the ribosomal RNA genes is as expected from the base composition of ribosomal RNA, others show a large discrepancy which cannot be due to the presence of low G-C spacer-DNA. The cross-hybridization observed with 1.3 and 0.7 × 106 molecular weight ribosomal RNAs and DNA, which varies greatly with different plant species, is not due to contamination of the ribosomal RNAs, and is specific for the ribosomal DNA of each species, probably largely restricted to those sequences coding for the two stable ribosomal RNAs. The double reciprocal plot may be used for the extrapolation of saturation values only with caution, because in these cases such plots are not linear over the whole of the hybridization reaction. PMID:16658392

  11. Apoptosis-induced mitochondrial dysfunction causes cytoplasmic lipid droplet formation.

    PubMed

    Boren, J; Brindle, K M

    2012-09-01

    A characteristic of apoptosis is the rapid accumulation of cytoplasmic lipid droplets, which are composed largely of neutral lipids. The proton signals from these lipids have been used for the non-invasive detection of cell death using magnetic resonance spectroscopy. We show here that despite an apoptosis-induced decrease in the levels and activities of enzymes involved in lipogenesis, which occurs downstream of p53 activation and inhibition of the mTOR signaling pathway, the increase in lipid accumulation is due to increased de novo lipid synthesis. This results from inhibition of mitochondrial fatty acid β-oxidation, which coupled with an increase in acyl-CoA synthetase activity, diverts fatty acids away from oxidation and into lipid synthesis. The inhibition of fatty acid oxidation can be explained by a rapid rise in mitochondrial membrane potential and an attendant increase in the levels of reactive oxygen species. PMID:22460322

  12. The Whereabouts of microRNA Actions: Cytoplasm and Beyond.

    PubMed

    Leung, Anthony K L

    2015-10-01

    MicroRNAs (miRNAs) are a conserved class of approximately 22 nucleotide (nt) short noncoding RNAs that normally silence gene expression via translational repression and/or degradation of targeted mRNAs in plants and animals. Identifying the whereabouts of miRNAs potentially informs miRNA functions, some of which are perhaps specialized to specific cellular compartments. In this review, the significance of miRNA localizations in the cytoplasm, including those at RNA granules and endomembranes, and the export of miRNAs to extracellular space will be discussed. How miRNA localizations and functions are regulated by protein modifications on the core miRNA-binding protein Argonaute (AGO) during normal and stress conditions will be explored, and in conclusion new AGO partners, non-AGO miRNA-binding proteins, and the emergent understanding of miRNAs found in the nucleoplasm, nucleoli, and mitochondria will be discussed. PMID:26410406

  13. Fighting an enemy within: cytoplasmic inhibitors of bacterial cysteine proteases.

    PubMed

    Potempa, Jan; Golonka, Ewa; Filipek, Renata; Shaw, Lindsey N

    2005-08-01

    The genes encoding secreted, broad-spectrum activity cysteine proteases of Staphylococcus spp. (staphopains) and Streptococcus pyogenes (streptopain, SpeB) are genetically linked to genes encoding cytoplasmic inhibitors. While staphopain inhibitors have lipocalin-like folds, streptopain is inhibited by a protein bearing the scaffold of the enzyme profragment. Bioinformatic analysis of other prokaryotic genomes has revealed that two more species may utilize this same genetic arrangement to control streptopain-like proteases with lipocalin-like inhibitors, while three other species may employ a C-terminally located domain that resembles the profragment. This apparently represents a novel system that bacteria use to control the intracellular activity of their proteases. PMID:16045606

  14. The Potassium Binding Protein Kbp Is a Cytoplasmic Potassium Sensor.

    PubMed

    Ashraf, Khuram U; Josts, Inokentijs; Mosbahi, Khedidja; Kelly, Sharon M; Byron, Olwyn; Smith, Brian O; Walker, Daniel

    2016-05-01

    Escherichia coli possesses a number of specific K(+) influx and efflux systems that maintain an appropriate intracellular K(+) concentration. Although regulatory mechanisms have been identified for a number of these transport systems, the exact mechanism through which K(+) concentration is sensed in the cell remains unknown. In this work we show that Kbp (K(+) binding protein, formerly YgaU), a soluble 16-kDa cytoplasmic protein from Escherichia coli, is a highly specific K(+) binding protein and is required for normal growth in the presence of high levels of external K(+). Kbp binds a single potassium ion with high specificity over Na(+) and other metal ions found in biological systems, although, in common with K(+) transporters, it also binds Rb(+) and Cs(+). Dissection of the K(+) binding determinants of Kbp suggests a mechanism through which Kbp is able to sense changes in K(+) concentration over the relevant range of intracellular K(+) concentrations. PMID:27112601

  15. Nucleo-cytoplasmic transport as a therapeutic target of cancer.

    PubMed

    Gravina, Giovanni Luca; Senapedis, William; McCauley, Dilara; Baloglu, Erkan; Shacham, Sharon; Festuccia, Claudio

    2014-01-01

    Shuttling of specific proteins out of the nucleus is essential for the regulation of the cell cycle and proliferation of both normal and malignant tissues. Dysregulation of this fundamental process may affect many other important cellular processes such as tumor growth, inflammatory response, cell cycle, and apoptosis. It is known that XPO1 (Exportin-1/Chromosome Region Maintenance 1/CRM1) is the main mediator of nuclear export in many cell types. Nuclear proteins exported to the cytoplasm by XPO1 include the drug targets topoisomerase IIα (topo IIα) and BCR-ABL and tumor suppressor proteins such as Rb, APC, p53, p21, and p27. XPO1 can mediate cell proliferation through several pathways: (i) the sub-cellular localization of NES-containing oncogenes and tumor suppressor proteins, (ii) the control of the mitotic apparatus and chromosome segregation, and (iii) the maintenance of nuclear and chromosomal structures. The XPO1 protein is elevated in ovarian carcinoma, glioma, osteosarcoma, pancreatic and cervical cancer. There is a growing body of research indicating that XPO1 may have an important role as a prognostic marker in solid tumors. Because of this, nuclear export inhibition through XPO1 is a potential target for therapeutic intervention in many cancers. The best understood XPO1 inhibitors are the small molecule nuclear export inhibitors (NEIs; Leptomycin B and derivatives, ratjadones, PKF050-638, valtrate, ACA, CBS9106, selinexor/KPT-330, and verdinexor/KPT-335). Selinexor and verdinexor are orally bioavailable, highly potent, small molecules that are classified as Selective Inhibitors of Nuclear Export (SINE). KPT-330 is the only NEI currently in Phase I/II human clinical trials in hematological and solid cancers. Of all the potential targets in nuclear cytoplasmic transport, the nuclear export receptor XPO1 remains the best understood and most advanced therapeutic target for the treatment of cancer. PMID:25476752

  16. Deep cytoplasmic rearrangements in axis-respecified Xenopus embryos

    NASA Technical Reports Server (NTRS)

    Denegre, J. M.; Danilchik, M. V.

    1993-01-01

    In fertilized eggs of the frog Xenopus, the vegetal yolk mass rotates away from the future dorsal side (J. P. Vincent and J. Gerhart, 1987, Dev. Biol. 123, 526-539), and a major rearrangement of the deep animal hemisphere cytoplasm produces a characteristic swirl in the prospective dorsal side (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). The relationship between this swirl and determination of the dorsal-ventral axis was further investigated by attempting to experimentally separate the positions of the swirl and the dorsal-ventral axis. Eggs were obliquely oriented in the gravity field to respecify the direction of yolk mass rotation and the position of the dorsal-ventral axis. When yolk mass rotation occurred in the absence of a sperm, as in activated eggs, a swirl pattern formed on the side away from which the yolk mass had rotated. In fertilized eggs tipped with the sperm entry point (SEP) down or to the side, swirl patterns were always found to form on the side away from which the yolk mass was displaced. However, in eggs tipped SEP up, in which the yolk mass was forced to rotate away from the SEP, more complicated rearrangements were observed in addition to the rotation-oriented swirl. Because the direction of yolk mass rotation was found to be influenced by both gravity and the actual position of the SEP in obliquely oriented eggs (SEP to the side), such complicated rearrangement patterns may result from opposing forces generated by both yolk mass rotation and the expanding sperm aster. Thus, except in cases in which the influences of SEP position and unit gravity opposed each other, it was not possible to experimentally separate the position of the deep cytoplasmic swirl from the direction of yolk mass rotation, and therefore the position of the prospective dorsal side.

  17. Cytoplasmic alkalization reduces calcium buffering in molluscan central neurons.

    PubMed

    Zucker, R S

    1981-11-23

    The effect of raised cytoplasmic pH (pHi) on intracellular concentration ([Ca2+]i) transients following calcium influx during membrane depolarization was studied in identified neurons in the abdominal ganglion of Aplysia californica. The pHi was monitored with pH-sensitive microelectrodes. Sea water containing 15 mM NH4Cl at pH 7.7 elevated pHi about 0.35 pH units from the normal level of 7.17. These cells have an estimated buffering power of about 60 mM/pH unit. Calcium influx was elicited by depolarizing pulses under voltage clamp and [Ca2+]i transients were monitored with the photoprotein aequorin or the metallochromic dye arsenazo III. Aequorin photo-emissions increased by 21--131% (mean, 48%) and arsenazo III absorbance changes accompanying depolarization increased by 9--33% (mean, 20%) after 30 min in NH4+, corresponding roughly to a 14% increase in [Ca2+]i transients. Calcium-dependent potassium tail currents following a depolarizing pulse were somewhat slower and 4--91% (mean, 38%) large in NH4+. The magnitude and time- and voltage-dependence of the membrane calcium conductance was studied using calcium tail currents following depolarizing pulses. The calcium current was unaffected by NH4+, so the enhanced [Ca2+]i transients must reflect reduced calcium buffering at high pHi. Either reduced cytoplasmic calcium binding or slowed active extrusion of calcium may be responsible for this effect. PMID:6271335

  18. Cytoplasmic genetic variation and extensive cytonuclear interactions influence natural variation in the metabolome

    PubMed Central

    Joseph, Bindu; Corwin, Jason A; Li, Baohua; Atwell, Suzi; Kliebenstein, Daniel J

    2013-01-01

    Understanding genome to phenotype linkages has been greatly enabled by genomic sequencing. However, most genome analysis is typically confined to the nuclear genome. We conducted a metabolomic QTL analysis on a reciprocal RIL population structured to examine how variation in the organelle genomes affects phenotypic variation. This showed that the cytoplasmic variation had effects similar to, if not larger than, the largest individual nuclear locus. Inclusion of cytoplasmic variation into the genetic model greatly increased the explained phenotypic variation. Cytoplasmic genetic variation was a central hub in the epistatic network controlling the plant metabolome. This epistatic influence manifested such that the cytoplasmic background could alter or hide pairwise epistasis between nuclear loci. Thus, cytoplasmic genetic variation plays a central role in controlling natural variation in metabolomic networks. This suggests that cytoplasmic genomes must be included in any future analysis of natural variation. DOI: http://dx.doi.org/10.7554/eLife.00776.001 PMID:24150750

  19. [Possible role of the cytoplasm in the course of morphogenesis, namely, in the case of twinning].

    PubMed

    Giroud, A

    1975-01-01

    A number of facts that cannot be interpreted in terms of nuclear genes would seem to be interpretable in terms of cytoplasmic heredity. The hereditary role of the mitochondria has already been demonstrated in molds. The role of the cytoplasm (matrilineal heredity) has also been shown in some phanerogams, and analogous facts have been noted in insects and molluscs. In amphibians, the influence of an alteration of the egg cortical cytoplasm has been shown to reappear in the following generations. This cortical cytoplasm includes the morphological plan of the organism with its bilateral symmetry. In Tatusia novemcincta the twins may be morphologically or chemically different, which may only be explained by an unequal subdivision of a heterogenic cytoplasm. Similar facts are observed in human twinning. Monozygotic twins are usually discordant with respect to congenital malformations (especially symmelia and anencephaly), which may only be interpreted in terms of unequal distribution of cytoplasmic properties. PMID:1242075

  20. A Phosphorylated Cytoplasmic Autoantigen, GW182, Associates with a Unique Population of Human mRNAs within Novel Cytoplasmic Speckles

    PubMed Central

    Eystathioy, Theophany; Chan, Edward K. L.; Tenenbaum, Scott A.; Keene, Jack D.; Griffith, Kevin; Fritzler, Marvin J.

    2002-01-01

    A novel human cellular structure has been identified that contains a unique autoimmune antigen and multiple messenger RNAs. This complex was discovered using an autoimmune serum from a patient with motor and sensory neuropathy and contains a protein of 182 kDa. The gene and cDNA encoding the protein indicated an open reading frame with glycine-tryptophan (GW) repeats and a single RNA recognition motif. Both the patient's serum and a rabbit serum raised against the recombinant GW protein costained discrete cytoplasmic speckles designated as GW bodies (GWBs) that do not overlap with the Golgi complex, endosomes, lysosomes, or peroxisomes. The mRNAs associated with GW182 represent a clustered set of transcripts that are presumed to reside within the GW complexes. We propose that the GW ribonucleoprotein complex is involved in the posttranscriptional regulation of gene expression by sequestering a specific subset of gene transcripts involved in cell growth and homeostasis. PMID:11950943

  1. Identification of cytoplasm types in rapeseed (Brassica napus L.) accessions by a multiplex PCR assay.

    PubMed

    Zhao, H X; Li, Z J; Hu, S W; Sun, G L; Chang, J J; Zhang, Z H

    2010-08-01

    Cytoplasmic male sterility (CMS) has widely been used as an efficient pollination control system in rapeseed hybrid production. Identification of cytoplasm type of rapeseed accessions is becoming the most important basic work for hybrid-rapeseed breeding. In this study, we report a simple multiplex PCR method to distinguish the existing common cytoplasm resources, Pol, Nap, Cam, Ogu and Ogu-NWSUAF cytoplasm, in rapeseed. Cytoplasm type of 35 F(1) hybrids and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested by this method. The results indicated that 10 of 35 F(1) hybrids are the Nap, and 25 the Pol cytoplasm type, which is consistent with the information provided by the breeders. Out of 140 accessions tested, 100 (71.4%), 21 (15%) and 19 (13.6%) accessions possess Nap, Cam and Pol cytoplasm, respectively. All 19 accessions with Pol cytoplasm are from China. Pedigree analysis indicated that these accessions with Pol cytoplasm were either restorers for Pol CMS, including Shaan 2C, Huiyehui, 220, etc. or derived from hybrids with Pol CMS as female parent. Our molecular results are consistent with those of the classical testcross, suggesting the reliability of this method. The multiplex PCR assay method can be applied to CMS "three-line" breeding, selection and validation of hybrid rapeseed. PMID:20401459

  2. Amphibian egg cytoplasm response to altered g-forces and gravity orientation

    NASA Technical Reports Server (NTRS)

    Neff, A. W.; Smith, R. C.; Malacinski, G. M.

    1986-01-01

    Elucidation of dorsal/ventral polarity and primary embryonic axis development in amphibian embryos requires an understanding of cytoplasmic rearrangements in fertile eggs at the biophysical, physiological, and biochemical levels. Evidence is presented that amphibian egg cytoplasmic components are compartmentalized. The effects of altered orientation to the gravitational vector (i.e., egg inversion) and alterations in gravity force ranging from hypergravity (centrifugation) to simulated microgravity (i.e., horizontal clinostat rotation) on cytoplasmic compartment rearrangements are reviewed. The behavior of yolk compartments as well as a newly defined (with monoclonal antibody) nonyolk cytoplasmic compartment, in inverted eggs and in eggs rotated on horizontal clinostats at their buoyant density, is discussed.

  3. Hindered Diffusion of Inert Tracer Particles in the Cytoplasm of Mouse 3T3 Cells

    NASA Astrophysics Data System (ADS)

    Luby-Phelps, Katherine; Castle, Philip E.; Lansing Taylor, D.; Lanni, Frederick

    1987-07-01

    Using fluorescence recovery after photobleaching, we have studied the diffusion of fluorescein-labeled, size-fractionated Ficoll in the cytoplasmic space of living Swiss 3T3 cells as a probe of the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier experiments with fluorescein-labeled, size-fractionated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dependent manner. Extrapolation of the data suggests that particles larger than 260 angstrom in radius may be completely nondiffusible in the cytoplasmic space. In contrast, diffusion of Ficoll in protein solutions of concentration comparable to the range reported for cytoplasm is not hindered in a size-dependent manner. Although we cannot at present distinguish among several physical chemical models for the organization of cytoplasm, these results make it clear that cytoplasm possesses some sort of higher-order intermolecular interactions (structure) not found in simple aqueous protein solutions, even at high concentration. These results also suggest that, for native cytoplasmic particles whose smallest radial dimension approaches 260 angstrom, size may be as important a determinant of cytoplasmic diffusibility as binding specificity. This would include most endosomes, polyribosomes, and the larger multienzyme complexes.

  4. Isolation of Cytoplasmic Pituitary Granules with Gonadotropic Activity

    PubMed Central

    Hartley, Marshall W.; McShan, W. H.; Ris, Hans

    1960-01-01

    A fraction isolated from the anterior pituitary glands of rats castrate for 8 weeks contained essentially a single cytoplasmic constituent with which the major portion of the gonadotropic hormone activity was associated. The glands were homogenized in an 0.25 M sucrose + 7.3 per cent polyvinylpyrrolidone (PVP) solution and fractionated by differential centrifugation to give a heterogeneous small granule fraction which contained almost all the gonadotropic hormone activity. The active supernatant containing this small granule fraction was separated into layers by isopycnic gradient centrifugation on a continuous 6 to 45 per cent sucrose + 17.5 per cent "diodrast" + 5 x 10-4 M "versene" gradient at 100,000 g for 2 hours. Three layers were obtained and the pellet from the active bottom layer was sectioned, examined with the electron microscope, and found to contain 200 mµ granules, mitochondria, ergastoplasm, and other cellular debris. This layer was fractionated further by isopycnic and differential centrifugation to obtain a pellet which contained the major portion of the gonadotropic hormone activity. Because of the heterogeneity of this fraction, due to the contamination of the 200 mµ granules with mitochondria and other cellular debris, the active layer and the resuspended active pellet, obtained by centrifuging this layer first at 17,000 g then diluting the supernatant and centrifuging at 30,000 g for 1 hour, were filtered through Millipore HA paper with a pore size of 0.45 µ. The cytoplasmic material containing the gonadotropic hormone activity passed through the filter paper and this activity was recovered in the pellets obtained by centrifuging at 100,000 g for 1 hour. These active pellets consisted almost entirely of 200 mµ granules with a minimum amount of contamination, and they contained the major portion of the gonadotropic hormone activity with practically none remaining in the supernatant fraction. These results are discussed in view of their

  5. Effects of alien and intraspecies cytoplasms on manifestation of nuclear genes for wheat resistance to brown rust: II. Specificity of cytoplasm influence on different Lr genes

    SciTech Connect

    Voluevich, E.A.; Buloichik, A.A.; Palilova, A.N.

    1995-04-01

    Specificity of expression of the major nuclear genes Lr to two brown rust clones in hybrids with the same maternal cytoplasm was analyzed. It was evaluated by a resistant: susceptible ratio in the F{sub 2}. Reciprocal hybrids were obtained from the cross between the progeny of homozygous susceptible plants of the cultivar Penjamo 62 and its alloplasmatic lines carrying cytoplasms of Triticum dicoccoides var. fulvovillosum, Aegilops squarrosa var. typical, Agropyron trichophorum, and isogenic lines of the cultivar Thatcher (Th) with the Lr1, Lr9, Lr15, and Lr19 genes. It was shown that the effect of the Lr1 gene in the cytoplasm of cultivar Thatcher and in eu-, and alloplasmatic forms of Penjamo 62 was less expressed than that of other Lr genes. Cytoplasm of the alloplasmatic line (dicoccoides)-Penjamo 62 was the only exception: in the F{sub 2}, hybrids with Th (Lr1) had a higher yield of resistant forms than those with Th (Lr15). In the hybrid combinations studied, expression and/or transmission of the Lr19 gene was more significant than that of other genes. This gene had no advantages over Lr15 and Lr19 only in cytoplasm of the alloplasmatic line (squarrosa)-Penjamo 62. In certain hybrid cytoplasms, the display of the Lr1, Lr15, and Lr19 genes, in contrast to Lr9, varied with the virulence of the pathogen clones. 15 refs., 5 tabs.

  6. Responses of alloplasmic (cytoplasm=Triticum timopheevii) and euplasmic wheats (Triticum aestivum) to photoperiod and vernalization.

    PubMed

    Ward, R W; Heyne, E G; Paulsen, G M

    1983-07-01

    Studies were conducted to determine the influence of the male sterility-inducing cytoplasm of Triticum timopheevii (Zhuk.) Zhuk. on response of several common winter wheat (T. aestivum L.) nuclear genotypes to photoperiod and vernalization. Comparative studies of cytoplasmic substitution lines provide information on the role of the cytoplasmic genetic mechanism in growth and development. In the case of cytoplasmic male sterility-based hybrid production systems, ubiquity of sterility-inducing cytoplasm in derived hybrids warrants thorough characterization of its influence on plant phenotype. Factorial combinations of cytoplasm (T. timopheevii and T. aestivum), nuclear genotype, and photoperiod or vernalization treatments were evaluated under hydroponic conditions in controlled environment chambers. Interaction of cytoplasm, photoperiod, and nuclear genotype was significant in one or more experiments for days to anthesis and potential spikelet number, and interaction of cytoplasm, vernalization, and nuclear genotype was significant for days to spike emergence. Long day length was associated with increased percentage seed set in one study, but interactions of photoperiod and cytoplasm were not detected for percentage seed set. Interactions involving cytoplasm and photoperiod or vernalization were interpreted as evidence of the existence of genetic factors in cytoplsam of T. timopheevii which alter photoperiod or vernalization responses of alloplasmic plants relative to responses exhibited by euplasmic plants. Since photoperiod and vernalization responses are critical to adaptation, T. timopheevii cytoplasm can alter adaptability of T. aestivum. The specific effect would be nuclear genotype dependent, and does not appear to be of a magnitude greater than that induced by nuclear genetic variability at loci conditioning photoperiod or vernalization responses or other adaptation-determining characteristics. Normal multilocation/year testing of alloplasmic hybrids should

  7. Lipogenic Enzymes Complexes and Cytoplasmic Lipid Droplet Formation During Adipogenesis.

    PubMed

    Padilla-Benavides, Teresita; Velez-delValle, Cristina; Marsch-Moreno, Meytha; Castro-Muñozledo, Federico; Kuri-Harcuch, Walid

    2016-10-01

    Lipid droplets are dynamic organelles that store triglycerides and participate in their mobilization in adipose cells. These organelles require the reorganization of some structural components, the cytoskeleton, and the activation of lipogenic enzymes. Using confocal microscopy, we analyzed the participation of cytoskeletal components and two lipogenic enzymes, fatty acid synthase and glycerophosphate dehydrogenase, during lipid droplet biogenesis in differentiating 3T3-F442A cells into adipocytes. We show that subcortical actin microfilaments are extended at the basal side of the cells in parallel arrangement to the culture dish substrate, and that the microtubule network traverses the cytoplasm as a scaffold that supports the round shape of the mature adipocyte. By immunoprecipitation, we show that vimentin and perilipin1a associate during the early stages of the differentiation process for lipid droplet formation. We also report that the antibody against perilipin1 detected a band that might correspond to a modified form of the molecule. Finally, the cytosolic distribution and punctate organization of lipogenic enzymes and their co-localization in the proximity of lipid droplets suggest the existence of dynamic protein complexes involved in synthesis and storage of triglycerides. J. Cell. Biochem. 117: 2315-2326, 2016. © 2016 Wiley Periodicals, Inc. PMID:26928794

  8. Cargo transport by cytoplasmic Dynein can center embryonic centrosomes.

    PubMed

    Longoria, Rafael A; Shubeita, George T

    2013-01-01

    To complete meiosis II in animal cells, the male DNA material needs to meet the female DNA material contained in the female pronucleus at the egg center, but it is not known how the male pronucleus, deposited by the sperm at the periphery of the cell, finds the cell center in large eggs. Pronucleus centering is an active process that appears to involve microtubules and molecular motors. For small and medium-sized cells, the force required to move the centrosome can arise from either microtubule pushing on the cortex, or cortically-attached dynein pulling on microtubules. However, in large cells, such as the fertilized Xenopus laevis embryo, where microtubules are too long to support pushing forces or they do not reach all boundaries before centrosome centering begins, a different force generating mechanism must exist. Here, we present a centrosome positioning model in which the cytosolic drag experienced by cargoes hauled by cytoplasmic dynein on the sperm aster microtubules can move the centrosome towards the cell's center. We find that small, fast cargoes (diameter ∼100 nm, cargo velocity ∼2 µm/s) are sufficient to move the centrosome in the geometry of the Xenopus laevis embryo within the experimentally observed length and time scales. PMID:23840877

  9. Association of Endophilin B1 with Cytoplasmic Vesicles.

    PubMed

    Li, Jinhui; Barylko, Barbara; Eichorst, John P; Mueller, Joachim D; Albanesi, Joseph P; Chen, Yan

    2016-08-01

    Endophilins are SH3- and BAR domain-containing proteins implicated in membrane remodeling and vesicle formation. Endophilins A1 and A2 promote the budding of endocytic vesicles from the plasma membrane, whereas endophilin B1 has been implicated in vesicle budding from intracellular organelles, including the trans-Golgi network and late endosomes. We previously reported that endophilins A1 and A2 exist almost exclusively as soluble dimers in the cytosol. Here, we present results of fluorescence fluctuation spectroscopy analyses indicating that, in contrast, the majority of endophilin B1 is present in multiple copies on small, highly mobile cytoplasmic vesicles. Formation of these vesicles was enhanced by overexpression of wild-type dynamin 2, but suppressed by expression of a catalytically inactive dynamin 2 mutant. Using dual-color heterospecies partition analysis, we identified the epidermal growth factor receptor on endophilin B1 vesicles. Moreover, a proportion of endophilin B1 vesicles also contained caveolin, whereas clathrin was almost undetectable on those vesicles. These results raise the possibility that endophilin B1 participates in dynamin 2-dependent formation of a population of transport vesicles distinct from those generated by A-type endophilins. PMID:27508440

  10. Novel clinical and diagnostic aspects of antineutrophil cytoplasmic antibodies.

    PubMed

    Schulte-Pelkum, Johannes; Radice, Antonella; Norman, Gary L; Lόpez Hoyos, Marcos; Lakos, Gabriella; Buchner, Carol; Musset, Lucile; Miyara, Makoto; Stinton, Laura; Mahler, Michael

    2014-01-01

    Antineutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of some idiopathic systemic vasculitides. Besides the investigation of ANCA-associated vasculitis (AAV) and constant effort for a standardized nomenclature and classification of the AAV, a main focus of research during the last few years has been to constantly improve the performance of enzyme immunoassays. With the latest so called third generation ELISA, this goal seemed to be fulfilled. The International Consensus Statement on Testing and Reporting of ANCA gave recommendations for standardized strategies for the serological diagnosis of ANCA. New developments now target the system immanent drawbacks of the respective diagnostic methods, be it the need for batching and the long time to result for ELISA, or the high likelihood of error and subjectivity of indirect immunofluorescence (IIF). Random access technology and multiplexing for solid phase assays as well as digital imaging for IIF are tools which may help to expedite and simplify routine diagnostics in the lab and in emergency settings. Recent findings indicate that PR3-ANCA have clinical utility beyond the diagnosis of AAV. PR3-ANCA can also serve as an aid for the differentiation between ulcerative colitis (UC) and Crohn's disease (CrD) and the stratification of UC patients. This review provides a detailed review of what is known about ANCA and highlights the latest research and state-of-the-art developments in this area. PMID:24995343

  11. Novel Clinical and Diagnostic Aspects of Antineutrophil Cytoplasmic Antibodies

    PubMed Central

    Schulte-Pelkum, Johannes; Radice, Antonella; Norman, Gary L.; Lόpez Hoyos, Marcos; Buchner, Carol; Musset, Lucile; Miyara, Makoto

    2014-01-01

    Antineutrophil cytoplasmic antibodies (ANCA) are the serological hallmark of some idiopathic systemic vasculitides. Besides the investigation of ANCA-associated vasculitis (AAV) and constant effort for a standardized nomenclature and classification of the AAV, a main focus of research during the last few years has been to constantly improve the performance of enzyme immunoassays. With the latest so called third generation ELISA, this goal seemed to be fulfilled. The International Consensus Statement on Testing and Reporting of ANCA gave recommendations for standardized strategies for the serological diagnosis of ANCA. New developments now target the system immanent drawbacks of the respective diagnostic methods, be it the need for batching and the long time to result for ELISA, or the high likelihood of error and subjectivity of indirect immunofluorescence (IIF). Random access technology and multiplexing for solid phase assays as well as digital imaging for IIF are tools which may help to expedite and simplify routine diagnostics in the lab and in emergency settings. Recent findings indicate that PR3-ANCA have clinical utility beyond the diagnosis of AAV. PR3-ANCA can also serve as an aid for the differentiation between ulcerative colitis (UC) and Crohn's disease (CrD) and the stratification of UC patients. This review provides a detailed review of what is known about ANCA and highlights the latest research and state-of-the-art developments in this area. PMID:24995343

  12. Excreted Cytoplasmic Proteins Contribute to Pathogenicity in Staphylococcus aureus.

    PubMed

    Ebner, Patrick; Rinker, Janina; Nguyen, Minh Thu; Popella, Peter; Nega, Mulugeta; Luqman, Arif; Schittek, Birgit; Di Marco, Moreno; Stevanovic, Stefan; Götz, Friedrich

    2016-06-01

    Excretion of cytoplasmic proteins in pro- and eukaryotes, also referred to as "nonclassical protein export," is a well-known phenomenon. However, comparatively little is known about the role of the excreted proteins in relation to pathogenicity. Here, the impact of two excreted glycolytic enzymes, aldolase (FbaA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), on pathogenicity was investigated in Staphylococcus aureus Both enzymes bound to certain host matrix proteins and enhanced adherence of the bacterial cells to host cells but caused a decrease in host cell invasion. FbaA and GAPDH also bound to the cell surfaces of staphylococcal cells by interaction with the major autolysin, Atl, that is involved in host cell internalization. Surprisingly, FbaA showed high cytotoxicity to both MonoMac 6 (MM6) and HaCaT cells, while GAPDH was cytotoxic only for MM6 cells. Finally, the contribution of external FbaA and GAPDH to S. aureus pathogenicity was confirmed in an insect infection model. PMID:27001537

  13. Cytoplasmic sphingosine-1-phosphate pathway modulates neuronal autophagy

    PubMed Central

    Moruno Manchon, Jose Felix; Uzor, Ndidi-Ese; Dabaghian, Yuri; Furr-Stimming, Erin E.; Finkbeiner, Steven; Tsvetkov, Andrey S.

    2015-01-01

    Autophagy is an important homeostatic mechanism that eliminates long-lived proteins, protein aggregates and damaged organelles. Its dysregulation is involved in many neurodegenerative disorders. Autophagy is therefore a promising target for blunting neurodegeneration. We searched for novel autophagic pathways in primary neurons and identified the cytosolic sphingosine-1-phosphate (S1P) pathway as a regulator of neuronal autophagy. S1P, a bioactive lipid generated by sphingosine kinase 1 (SK1) in the cytoplasm, is implicated in cell survival. We found that SK1 enhances flux through autophagy and that S1P-metabolizing enzymes decrease this flux. When autophagy is stimulated, SK1 relocalizes to endosomes/autophagosomes in neurons. Expression of a dominant-negative form of SK1 inhibits autophagosome synthesis. In a neuron model of Huntington’s disease, pharmacologically inhibiting S1P-lyase protected neurons from mutant huntingtin-induced neurotoxicity. These results identify the S1P pathway as a novel regulator of neuronal autophagy and provide a new target for developing therapies for neurodegenerative disorders. PMID:26477494

  14. Downy mildew incidence of pearl millet hybrids with different male-sterility inducing cytoplasms.

    PubMed

    Yadav, O P

    1996-02-01

    The use of different sources of cytoplasmic male sterility (CMS) in hybrid seed production of pearl millet [Pennisetum glaucum (L.) R. Br.] is advocated to avoid possible disease epidemics occurring due to cytoplasmic uniformity. The effects of commercially unexploited, but potentially exploitable, sources of CMS, like A2, A3 and A4, on downy mildew [Sclerospora graminicola (Sacc.) Schroet] incidence were studied by using the disease incidence of isonuclear hybrids with male-sterile and fertile cytoplasm. The mean downy mildew incidence of hybrids carrying different male-sterile cytoplasm was similar to that of hybrids retaining the fertile cytoplasm. The cytoplasm accounted for only 0.6% of the total variation and its effect was non-significant; pollinators could explain most of the variation in determining the disease incidence of hybrids. This suggested that these male-sterile cytoplasms are not linked to downy mildew susceptibility and thus can be exploited commercially to broaden the cytoplasmic base of the male-sterile lines and, ultimately, of hybrids. PMID:24166178

  15. Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

    PubMed

    Sawatsky, Bevan; Bente, Dennis A; Czub, Markus; von Messling, Veronika

    2016-05-01

    The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle. PMID:26813519

  16. TRIM5{alpha} association with cytoplasmic bodies is not required for antiretroviral activity

    SciTech Connect

    Song, Byeongwoon; Diaz-Griffero, Felipe; Park, Do Hyun; Rogers, Thomas; Stremlau, Matthew; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2005-12-20

    The tripartite motif (TRIM) protein, TRIM5{alpha}, restricts infection by particular retroviruses. Many TRIM proteins form cytoplasmic bodies of unknown function. We investigated the relationship between cytoplasmic body formation and the structure and antiretroviral activity of TRIM5{alpha}. In addition to diffuse cytoplasmic staining, the TRIM5{alpha} proteins from several primate species were located in cytoplasmic bodies of different sizes; by contrast, TRIM5{alpha} from spider monkeys did not form cytoplasmic bodies. Despite these differences, all of the TRIM5{alpha} proteins exhibited the ability to restrict infection by particular retroviruses. Treatment of cells with geldanamycin, an Hsp90 inhibitor, resulted in disappearance or reduction of the TRIM5{alpha}-associated cytoplasmic bodies, yet exerted little effect on the restriction of retroviral infection. Studies of green fluorescent protein-TRIM5{alpha} fusion proteins indicated that no TRIM5{alpha} domain is specifically required for association with cytoplasmic bodies. Apparently, the formation of cytoplasmic bodies is not required for the antiretroviral activity of TRIM5{alpha}.

  17. Cytoplasmic pathway followed by chloride ions to enter the CFTR channel pore.

    PubMed

    El Hiani, Yassine; Negoda, Alexander; Linsdell, Paul

    2016-05-01

    Most ATP-binding cassette (ABC) proteins function as ATP-dependent membrane pumps. One exception is the cystic fibrosis transmembrane conductance regulator (CFTR), an ABC protein that functions as a Cl(-) ion channel. As such, the CFTR protein must form a continuous pathway for the movement of Cl(-) ions from the cytoplasm to the extracellular solution when in its open channel state. Extensive functional investigations have characterized most parts of this Cl(-) permeation pathway. However, one region remains unexplored-the pathway connecting the cytoplasm to the membrane-spanning pore. We used patch clamp recording and extensive substituted cysteine accessibility mutagenesis to identify amino acid side-chains in cytoplasmic regions of CFTR that lie close to the pathway taken by Cl(-) ions as they pass from the cytoplasm through this pathway. Our results suggest that Cl(-) ions enter the permeation pathway via a single lateral tunnel formed by the cytoplasmic parts of the protein, and then follow a fairly direct central pathway towards the membrane-spanning parts of the protein. However, this pathway is not lined continuously by any particular part of the protein; instead, the contributions of different cytoplasmic regions of the protein appear to change as the permeation pathway approaches the membrane, which appears to reflect the ways in which different cytoplasmic regions of the protein are oriented towards its central axis. Our results allow us to define for the first time the complete Cl(-) permeation pathway in CFTR, from the cytoplasm to the extracellular solution. PMID:26659082

  18. Effect of wild Helianthus cytoplasms on agronomic and oil characteristics of cultivated sunflower (H. annuus L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sunflower (Helianthus annuus L.) productions reliance on a single source of cytoplasmic male-sterility, PET1, derived from H. petiolaris Nutt., makes the crop genetically vulnerable. Twenty diverse cytoplasmic substitution lines from annual and perennial wild species were compared with the inbred li...

  19. Cytoplasmic Rbfox1 Regulates the Expression of Synaptic and Autism-Related Genes.

    PubMed

    Lee, Ji-Ann; Damianov, Andrey; Lin, Chia-Ho; Fontes, Mariana; Parikshak, Neelroop N; Anderson, Erik S; Geschwind, Daniel H; Black, Douglas L; Martin, Kelsey C

    2016-01-01

    Human genetic studies have identified the neuronal RNA binding protein, Rbfox1, as a candidate gene for autism spectrum disorders. While Rbfox1 functions as a splicing regulator in the nucleus, it is also alternatively spliced to produce cytoplasmic isoforms. To investigate the function of cytoplasmic Rbfox1, we knocked down Rbfox proteins in mouse neurons and rescued with cytoplasmic or nuclear Rbfox1. Transcriptome profiling showed that nuclear Rbfox1 rescued splicing changes, whereas cytoplasmic Rbfox1 rescued changes in mRNA levels. iCLIP-seq of subcellular fractions revealed that Rbfox1 bound predominantly to introns in nascent RNA, while cytoplasmic Rbox1 bound to 3' UTRs. Cytoplasmic Rbfox1 binding increased target mRNA stability and translation, and Rbfox1 and miRNA binding sites overlapped significantly. Cytoplasmic Rbfox1 target mRNAs were enriched in genes involved in cortical development and autism. Our results uncover a new Rbfox1 regulatory network and highlight the importance of cytoplasmic RNA metabolism to cortical development and disease. PMID:26687839

  20. Response of amphibian egg non-yolk cytoplasm to gravity orientation

    NASA Technical Reports Server (NTRS)

    Smith, R. C.; Neff, A. W.; Malacinski, G. M.

    1985-01-01

    In order to study amphibian egg cytoplasmic organization and egg symmetrization at the molecular level, a library of seventeen monoclonal antibodies (MoAbs) against Xenopus laevis non-yolk egg proteins was produced. Several of these MoAbs react with non-yolk cytoplasmic antigens which are unevenly distributed in the fertile Xenopus egg.

  1. CYTOLOGY OF CANDIDA ALBICANS AS INFLUENCED BY DRUGS ACTING ON THE CYTOPLASMIC MEMBRANE

    PubMed Central

    Gale, Glen R.

    1963-01-01

    Gale, Glen R. (Veterans Administration Hospital, Durham, N.C.) Cytology of Candida albicans as influenced by drugs acting on the cytoplasmic membrane. J. Bacteriol. 86:151–157. 1963.—An electron microscopic comparison was made of the cytological responses of Candida albicans to benzalkonium chloride, amphotericin B, and filipin, all of which are thought to exert their primary pharmacological actions on the cytoplasmic membrane of susceptible microorganisms. After 5 to 15 min of exposure to benzalkonium chloride, most of the cytoplasm became less dense, and intact nuclei, mitochondria, or intracytoplasmic membranes were not observed. The remaining dense cytoplasm contained numerous small holes. The polyene antifungal agents, amphotericin B and filipin, caused a reduction in cytoplasmic density, but had no observable effect on nuclei or mitochondria. The intervals before onset of changes induced by the polyene agents correlated with the known time-dependent binding of these drugs by cells, and the decreased electron scattering induced by all three agents was compatible with their known abilities to cause a loss of integrity of the cytoplasmic membrane with resultant leakage of cytoplasmic components. No consistent morphological effect of any of these drugs on the cytoplasmic membrane could be shown. Images PMID:14054376

  2. A perspective on the mobilization, localization and delivery of molecules in the crowded bacterial cytoplasm.

    PubMed

    Trevors, J T

    2012-01-01

    It has been assumed that diffusion of molecules in the bacterial cytoplasm is the mechanism that moves molecules in the absence of cytoplasmic streaming. However, is there an undiscovered mechanism present that mobilizes cytoplasm and its molecular contents, and delivers tRNAs to specific ribosomes at specific bacterial cytoplasmic locations? Mobilization of specific tRNA (and also mRNA transcripts and ribosomes) and cell division proteins to specific intracellular locations may suggest that instructions and/or mechanism(s) are needed. The alternative is that molecular crowding in the cytoplasm is sufficient for gentle contact between mRNA, ribosomes and tRNA. Or is it plausible that the bacterial cytoplasm (and its contents) are mobilized with the outcome being more gentle collisions between molecules than by a diffusion only mechanism? One hypothesis is that cytoplasmic and molecule mobilization and spatial organization are possibly driven by the photons in thermal infrared (IR) radiation and generation of exclusion zone (EZ) water in the cytoplasm. PMID:22086198

  3. Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria

    PubMed Central

    Robinson, Michael-Paul; Ke, Na; Lobstein, Julie; Peterson, Cristen; Szkodny, Alana; Mansell, Thomas J.; Tuckey, Corinna; Riggs, Paul D.; Colussi, Paul A.; Noren, Christopher J.; Taron, Christopher H.; DeLisa, Matthew P.; Berkmen, Mehmet

    2015-01-01

    Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs—named ‘cyclonals'—effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. PMID:26311203

  4. Cytoplasmic dynein is a minus end-directed motor for membranous organelles

    SciTech Connect

    Schroer, T.A.; Steuer, E.R.; Sheetz, M.P.

    1989-03-24

    The role of cytoplasmic dynein in microtubule-based organelle transport was examined using a reconstituted assay developed from chick embryo fibroblasts. Factors present in a high-speed cytosol caused the movement of purified organelles on microtubules predominantly in the minus end direction. Inactivation of cytoplasmic dynein in the high-speed cytosol by vanadate-mediated UV photocleavage inhibited minus end-directed organelle motility by over 90%. Addition of purified cytoplasmic dynein to the inactive cytosol restored minus end-directed organelle motility, although purified cytoplasmic dynein by itself did not support organelle movement. We propose that cytoplasmic dynein is the motor for minus end-directed organelle movement, but that additional cytosolic factors are also required to produce organelle motility.

  5. Cytoplasmic membrane response to copper and nickel in Acidithiobacillus ferrooxidans.

    PubMed

    Mykytczuk, N C S; Trevors, J T; Ferroni, G D; Leduc, L G

    2011-03-20

    Metal tolerance has been found to vary among Acidithiobacillus ferrooxidans strains and this can impact the efficiency of biomining practices. To explain observed strain variability for differences in metal tolerance we examined the effects of Cu(2+) and Ni(2+) concentrations (1-200 mM) on cytoplasmic membrane properties of two A. ferrooxidans type strains (ATCC 23270 and 19859) and four strains isolated from AMD water around Sudbury, Ontario, Canada. Growth rate, membrane fluidity and phase, determined from the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), and fatty acid profiles indicated that three different modes of adaptation were present and could separate between strains showing moderate, or high metal tolerance from more sensitive strains. To compensate for the membrane ordering effects of the metals, significant remodelling of the membrane was used to either maintain homeoviscous adaptation in the moderately tolerant strains or to increase membrane fluidity in the sensitive strains. Shifts in the gel-to-liquid crystalline transition temperature in the moderately tolerant strains led to multiple phase transitions, increasing the potential for phase separation and compromised membrane integrity. The metal-tolerant strain however, was able to tolerate increases in membrane order without significant compensation via fatty acid composition. Our multivariate analyses show a common adaptive response which involves changes in the abundant 16:0 and 18:1 fatty acids. However, fatty acid composition and membrane properties showed no difference in response to either copper or nickel suggesting that adaptive response was non-specific and tolerance dependent. We demonstrate that strain variation can be evaluated using differences in membrane properties as intrinsic determinants of metal susceptibility. PMID:20630730

  6. Antineutrophil cytoplasmic autoantibody-associated glomerulonephritis in children.

    PubMed

    Hattori, M; Kurayama, H; Koitabashi, Y

    2001-07-01

    Aretrospective investigation was conducted by members of the Japanese Society for Pediatric Nephrology from 1990 to 1997 to define the clinical features and outcome of antineutrophil cytoplasmic autoantibody (ANCA)-associated glomerulonephritis in children. Thirty-four ANCA-seropositive Japanese pediatric patients with biopsy-proven pauci-immune necrotizing crescentic glomerulonephritis were identified. Of these, 3 cases associated with Wegener's granulomatosis were excluded because of the small sample size. Among the 31 patients studied, 10 had a diagnosis of necrotizing crescentic glomerulonephritis alone and 21 had microscopic polyangiitis. Females predominated (87%), and the median age at onset was 12 yr. Twenty-six patients received treatment with cyclophosphamide and corticosteroids, and five patients received treatment with corticosteroids alone; 84% of patients achieved remission, and 39% of responders relapsed in a median of 24 mo. ANCA titers correlated with response to treatment and disease activity, with some exceptions. Patients were followed for a median of 42 mo (range, 3 to 96 mo). Nine of 31 patients (29.0%) progressed to end-stage renal disease, 6 (19.4%) had reduced renal function, and 15 (48.4%) had normal renal function at the last observation. One patient (3.2%) died from cytomegalovirus infection 3 mo after initiation of therapy. Life-table analysis showed 75% renal survival at 39 mo. Patients who subsequently developed end-stage renal disease (n = 9) had significantly higher average peak serum creatinine levels and more chronic pathologic lesions at diagnosis compared with patients with favorable renal outcome (n = 15). In conclusion, our clinical experience suggests that the clinical disease spectrum of ANCA-associated glomerulonephritis is similar in pediatric and adult patients, but there is a female predominance in children. PMID:11423578

  7. Studying Genome Heterogeneity within the Arbuscular Mycorrhizal Fungal Cytoplasm

    PubMed Central

    Halary, Sébastien; Bapteste, Eric; Hijri, Mohamed

    2015-01-01

    Although heterokaryons have been reported in nature, multicellular organisms are generally assumed genetically homogeneous. Here, we investigate the case of arbuscular mycorrhizal fungi (AMF) that form symbiosis with plant roots. The growth advantages they confer to their hosts are of great potential benefit to sustainable agricultural practices. However, measuring genetic diversity for these coenocytes is a major challenge: Within the same cytoplasm, AMF contain thousands of nuclei and show extremely high levels of genetic variation for some loci. The extent and physical location of polymorphism within and between AMF genomes is unclear. We used two complementary strategies to estimate genetic diversity in AMF, investigating polymorphism both on a genome scale and in putative single copy loci. First, we used data from whole-genome pyrosequencing of four AMF isolates to describe genetic diversity, based on a conservative network-based clustering approach. AMF isolates showed marked differences in genome-wide diversity patterns in comparison to a panel of control fungal genomes. This clustering approach further allowed us to provide conservative estimates of Rhizophagus spp. genomes sizes. Second, we designed new putative single copy genomic markers, which we investigated by massive parallel amplicon sequencing for two Rhizophagus irregularis and one Rhizophagus sp. isolates. Most loci showed high polymorphism, with up to 103 alleles per marker. This polymorphism could be distributed within or between nuclei. However, we argue that the Rhizophagus isolates under study might be heterokaryotic, at least for the putative single copy markers we studied. Considering that genetic information is the main resource for identification of AMF, we suggest that special attention is warranted for the study of these ecologically important organisms. PMID:25573960

  8. Influence of A1 cytoplasmic substitution on the downy-mildew incidence of pearl millet.

    PubMed

    Yadav, O P; Manga, V K; Gupta, G K

    1993-12-01

    Large-scale cultivation of pearl millet [Pennisetum glaucum (L.) R. Br. F1 hybrids in India has led to increased incidence of downy-mildew (Sclerospora graminicola). There is concern that the A1 male-sterile cytoplasm used in all the hybrids released so far is responsible for this increase. The influence of A1 malesterile cytoplasm on downy-mildew incidence in pearl millet was studied by comparing the disease reaction of 40 pairs of F1 hybrids, each pair carrying respectively a1 male-sterile and normal B cytoplasm. Mean downy-mildew incidence was similar in the hybrids carrying either A1 male-sterile or B cytoplasm. The general combining ability of lines with and without A1 cytoplasm was found to be similar for downy-mildew incidence. These results indicated that in pearl millet A1 cytoplasm is not associated with increased downymildew incidence. The possible danger of using only one source of cytoplasm has been briefly discussed. PMID:24190349

  9. Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells.

    PubMed

    Kalantari, Roya; Hicks, Jessica A; Li, Liande; Gagnon, Keith T; Sridhara, Viswanadham; Lemoff, Andrew; Mirzaei, Hamid; Corey, David R

    2016-07-01

    Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners. PMID:27198507

  10. Cytoplasmic Acidification Induced by Inorganic Phosphate Uptake in Suspension Cultured Catharanthus roseus Cells

    PubMed Central

    Sakano, Katsuhiro; Yazaki, Yoshiaki; Mimura, Tetsuro

    1992-01-01

    Cytoplasmic acidification during inorganic phosphate (Pi) absorption by Catharanthus roseus cells were studied by means of a fluorescent pH indicator, 2′,7′-bis-(2-carboxyethyl)-5 carboxyfluorescein (acetomethylester) (BCECF), and 31P-nuclear magnetic resonance spectroscopy. Cytoplasmic acidification measured by decrease in the fluorescence intensity started immediately after Pi application. Within a minute or so, a stable state was attained and no further acidification occurred, whereas Pi absorption was still proceeding. As soon as Pi in the medium was exhausted, cytoplasmic pH started to recover. Coincidentally, the medium pH started to recover toward the original acidic pH. The Pi-induced changes in the cytoplasmic pH were confirmed by 31P-nuclear magnetic resonance study. Maximum acidification of the cytoplasm induced by 1.7 millimolar Pi was 0.2 pH units. Vacuolar pH was also affected by Pi. In some experiments, but not all, pH decreased reversibly by 0.2 to 0.3 pH units during Pi absorption. Results suggest that the cytoplasmic pH is regulated by proton pumps in the plasma membrane and in the tonoplast. In addition, other mechanisms that could consume extra protons in the cytoplasm are suggested. ImagesFigure 1 PMID:16668939

  11. Novel nuclear-cytoplasmic interaction in wheat (Triticum aestivum) induces vigorous plants.

    PubMed

    Soltani, Ali; Kumar, Ajay; Mergoum, Mohamed; Pirseyedi, Seyed Mostafa; Hegstad, Justin B; Mazaheri, Mona; Kianian, Shahryar F

    2016-03-01

    Interspecific hybridization can be considered an accelerator of evolution, otherwise a slow process, solely dependent on mutation and recombination. Upon interspecific hybridization, several novel interactions between nuclear and cytoplasmic genomes emerge which provide additional sources of diversity. The magnitude and essence of intergenomic interactions between nuclear and cytoplasmic genomes remain unknown due to the direction of many crosses. This study was conducted to address the role of nuclear-cytoplasmic interactions as a source of variation upon hybridization. Wheat (Triticum aestivum) alloplasmic lines carrying the cytoplasm of Aegilops mutica along with an integrated approach utilizing comparative quantitative trait locus (QTL) and epigenome analysis were used to dissect this interaction. The results indicate that cytoplasmic genomes can modify the magnitude of QTL controlling certain physiological traits such as dry matter weight. Furthermore, methylation profiling analysis detected eight polymorphic regions affected by the cytoplasm type. In general, these results indicate that novel nuclear-cytoplasmic interactions can potentially trigger an epigenetic modification cascade in nuclear genes which eventually change the genetic network controlling physiological traits. These modified genetic networks can serve as new sources of variation to accelerate the evolutionary process. Furthermore, this variation can synthetically be produced by breeders in their programs to develop epigenomic-segregating lines. PMID:26860316

  12. Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells

    PubMed Central

    Kalantari, Roya; Hicks, Jessica A.; Li, Liande; Gagnon, Keith T.; Sridhara, Viswanadham; Lemoff, Andrew; Mirzaei, Hamid; Corey, David R.

    2016-01-01

    Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners. PMID:27198507

  13. Targeted Cytoplasmic Irradiation with Alpha Particles Induces Mutations in Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Wu, Li-Jun; Randers-Pehrson, Gerhard; Xu, An; Waldren, Charles A.; Geard, Charles R.; Yu, Zengliang; Hei, Tom K.

    1999-04-01

    Ever since x-rays were shown to induce mutation in Drosophila more than 70 years ago, prevailing dogma considered the genotoxic effects of ionizing radiation, such as mutations and carcinogenesis, as being due mostly to direct damage to the nucleus. Although there was indication that alpha particle traversal through cellular cytoplasm was innocuous, the full impact remained unknown. The availability of the microbeam at the Radiological Research Accelerator Facility of Columbia University made it possible to target and irradiate the cytoplasm of individual cells in a highly localized spatial region. By using dual fluorochrome dyes (Hoechst and Nile Red) to locate nucleus and cellular cytoplasm, respectively, thereby avoiding inadvertent traversal of nuclei, we show here that cytoplasmic irradiation is mutagenic at the CD59 (S1) locus of human-hamster hybrid (AL) cells, while inflicting minimal cytotoxicity. The principal class of mutations induced are similar to those of spontaneous origin and are entirely different from those of nuclear irradiation. Furthermore, experiments with radical scavenger and inhibitor of intracellular glutathione indicated that the mutagenicity of cytoplasmic irradiation depends on generation of reactive oxygen species. These findings suggest that cytoplasm is an important target for genotoxic effects of ionizing radiation, particularly radon, the second leading cause of lung cancer in the United States. In addition, cytoplasmic traversal by alpha particles may be more dangerous than nuclear traversal, because the mutagenicity is accomplished by little or no killing of the target cells.

  14. Single-molecule study of molecular mobility in the cytoplasm of Escherichia coli

    NASA Astrophysics Data System (ADS)

    Lill, Yoriko; Kaserer, Wallace A.; Newton, Salete M.; Lill, Markus; Klebba, Phillip E.; Ritchie, Ken

    2012-08-01

    The cytoplasm of bacterial cells is filled with individual molecules and molecular complexes that rely on diffusion to bring them together for interaction. The mobility of molecules in the cytoplasm has been characterized by several techniques mainly using fluorescent probes and ensemble methods. In order to probe the microenvrionment inside the cytoplasm as viewed by an individual molecule, we have studied single green fluorescent proteins (GFPs) diffusing in the cytoplasm of Escherichia coli cells at observation at rates ranging from 60 to 1000 Hz. Over long times the diffusion shows confinement due to the geometry of the cells themselves. A simulation in model cells using the actual distribution of cell sizes found in the experiments describes accurately the experimental results as well as reveals a short time diffusion coefficient that agrees well with that determined by ensemble methods. Higher short time diffusion coefficients can be obtained by filling the simulated cell with small spheres modeling cytoplasmic molecules and, depending on the density of particles included in the modeled cytoplasm, can approach the diffusion coefficient of GFPs found in water. Thus, single-molecule tracking combined with analysis using simple simulation of Brownian motion is able to reveal the main contributors to the GFP mobility in the cytoplasm of E. coli.

  15. Single-molecule study of molecular mobility in the cytoplasm of Escherichia coli.

    PubMed

    Lill, Yoriko; Kaserer, Wallace A; Newton, Salete M; Lill, Markus; Klebba, Phillip E; Ritchie, Ken

    2012-08-01

    The cytoplasm of bacterial cells is filled with individual molecules and molecular complexes that rely on diffusion to bring them together for interaction. The mobility of molecules in the cytoplasm has been characterized by several techniques mainly using fluorescent probes and ensemble methods. In order to probe the microenvrionment inside the cytoplasm as viewed by an individual molecule, we have studied single green fluorescent proteins (GFPs) diffusing in the cytoplasm of Escherichia coli cells at observation at rates ranging from 60 to 1000 Hz. Over long times the diffusion shows confinement due to the geometry of the cells themselves. A simulation in model cells using the actual distribution of cell sizes found in the experiments describes accurately the experimental results as well as reveals a short time diffusion coefficient that agrees well with that determined by ensemble methods. Higher short time diffusion coefficients can be obtained by filling the simulated cell with small spheres modeling cytoplasmic molecules and, depending on the density of particles included in the modeled cytoplasm, can approach the diffusion coefficient of GFPs found in water. Thus, single-molecule tracking combined with analysis using simple simulation of Brownian motion is able to reveal the main contributors to the GFP mobility in the cytoplasm of E. coli. PMID:23005785

  16. Pulmonary Fibrosis in Antineutrophil Cytoplasmic Antibodies (ANCA)-Associated Vasculitis

    PubMed Central

    Comarmond, Cloé; Crestani, Bruno; Tazi, Abdellatif; Hervier, Baptiste; Adam-Marchand, Sylvain; Nunes, Hilario; Cohen-Aubart, Fleur; Wislez, Marie; Cadranel, Jacques; Housset, Bruno; Lloret-Linares, Célia; Sève, Pascal; Pagnoux, Christian; Abad, Sébastien; Camuset, Juliette; Bienvenu, Boris; Duruisseaux, Michaël; Hachulla, Eric; Arlet, Jean-Benoît; Hamidou, Mohammed; Mahr, Alfred; Resche-Rigon, Matthieu; Brun, Anne-Laure; Grenier, Philippe; Cacoub, Patrice; Saadoun, David

    2014-01-01

    Abstract Pulmonary fibrosis (PF) is an uncommon manifestation observed in patients with antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV), particularly microscopic polyangiitis (MPA). While patients with PF associated with AAV seem to have a worse prognosis, these patients have been described only in case reports or small retrospective case series. In this retrospective multicenter study, we report the main features and long-term outcomes of patients with PF associated with AAV, fulfilling the American College of Rheumatology criteria and/or Chapel Hill definitions. Forty-nine patients (30 men [61%]; median age at diagnosis of AAV, 68 [interquartile range, 58–73] years) with PF associated with AAV were identified. Forty (81.6%) patients had MPA and 9 (18.4%) had granulomatosis with polyangiitis. The diagnosis of PF preceded the onset of vasculitis in 22 (45%) patients. Usual interstitial pneumonia was the main radiologic pattern (n = 18, 43%). ANCA were mostly of antimyeloperoxidase specificity (88%). All patients were treated with glucocorticoids as induction therapy, combined with cyclophosphamide (CYC) (n = 36, 73.5%) or rituximab (RTX) (n = 1, 2%). Factors associated with mortality included occurrence of chronic respiratory insufficiency (hazard ratio [HR], 7.44; 95% confidence interval [CI], 1.6–34.5; p = 0.003), induction therapy with glucocorticoids alone (HR, 2.94; CI, 1.05–8.33; p = 0.04), and initial weigh loss (HR, 2.83; CI, 1.05–7.65; p = 0.041). The 3-year survival rate in patients treated with glucocorticoids alone or combined with an immunosuppressant (CYC or RTX) as induction therapy was 64% (95% CI, 41–99) and 94% (95% CI, 86–100), respectively (p = 0.03). After a median follow-up of 48 months [interquartile range, 14–88 mo], 18 (37%) patients died, including 11 related to respiratory insufficiency. PF is a rare manifestation of AAV with a very poor prognosis. Induction therapy with

  17. Hypertrophic pachymeningitis: significance of myeloperoxidase anti-neutrophil cytoplasmic antibody.

    PubMed

    Yokoseki, Akiko; Saji, Etsuji; Arakawa, Musashi; Kosaka, Takayuki; Hokari, Mariko; Toyoshima, Yasuko; Okamoto, Kouichirou; Takeda, Shigeki; Sanpei, Kazuhiro; Kikuchi, Hirotoshi; Hirohata, Shunsei; Akazawa, Kouhei; Kakita, Akiyoshi; Takahashi, Hitoshi; Nishizawa, Masatoyo; Kawachi, Izumi

    2014-02-01

    The aim of this study was to elucidate the characteristics, pathogenesis and treatment strategy of hypertrophic pachymeningitis that is associated with myeloperoxidase anti-neutrophil cytoplasmic antibody (ANCA). We retrospectively investigated clinical, radiological, immunological and pathological profiles of 36 patients with immune-mediated or idiopathic hypertrophic pachymeningitis, including 17 patients with myeloperoxidase-ANCA, four patients with proteinase 3-ANCA, six patients with other immune-mediated disorders, and nine patients with 'idiopathic' variety. Myeloperoxidase-ANCA-positive hypertrophic pachymeningitis was characterized by: (i) an elderly female predominance; (ii) 82% of patients diagnosed with granulomatosis with polyangiitis (previously known as Wegener's granulomatosis) according to Watts' algorithm; (iii) a high frequency of patients with lesions limited to the dura mater and upper airways, developing headaches, chronic sinusitis, otitis media or mastoiditis; (iv) a low frequency of patients with the 'classical or generalized form' of granulomatosis with polyangiitis involving the entire upper and lower airways and kidney, or progressing to generalized disease, in contrast to proteinase 3-ANCA-positive hypertrophic pachymeningitis; (v) less severe neurological damage according to the modified Rankin Scale and low disease activity according to the Birmingham Vasculitis Activity Score compared with proteinase 3-ANCA-positive hypertrophic pachymeningitis; (vi) increased levels of CXCL10, CXCL8 and interleukin 6 in cerebrospinal fluids, and increased numbers of T cells, neutrophils, eosinophils, plasma cells and monocytes/macrophages in autopsied or biopsied dura mater with pachymeningitis, suggesting TH1-predominant granulomatous lesions in hypertrophic pachymeningitis, as previously reported in pulmonary or renal lesions of granulomatosis with polyangiitis; and (vii) greater efficacy of combination therapy with prednisolone and

  18. Cytoplasm segmentation on cervical cell images using graph cut-based approach.

    PubMed

    Zhang, Ling; Kong, Hui; Chin, Chien Ting; Wang, Tianfu; Chen, Siping

    2014-01-01

    This paper proposes a method to segment the cytoplasm in cervical cell images using graph cut-based algorithm. First, the A* channel in CIE LAB color space is extracted for contrast enhancement. Then, in order to effectively extract cytoplasm boundaries when image histograms present non-bimodal distribution, Otsu multiple thresholding is performed on the contrast enhanced image to generate initial segments, based on which the segments are refined by the multi-way graph cut method. We use 21 cervical cell images with non-ideal imaging condition to evaluate cytoplasm segmentation performance. The proposed method achieved a 93% accuracy which outperformed state-of-the-art works. PMID:24212005

  19. Cytoplasmic streaming affects gravity-induced amyloplast sedimentation in maize coleoptiles

    NASA Technical Reports Server (NTRS)

    Sack, F. D.; Leopold, A. C.

    1985-01-01

    Living maize (Zea mays L.) coleoptile cells were observed using a horizontal microscope to determine the interaction between cytoplasmic streaming and gravity-induced amyloplast sedimentation. Sedimentation is heavily influenced by streaming which may (1) hasten or slow the velocity of amyloplast movement and (2) displace the plastid laterally or even upwards before or after sedimentation. Amyloplasts may move through transvacuolar strands or through the peripheral cytoplasm which may be divided into fine cytoplasmic strands of much smaller diameter than the plastids. The results indicate that streaming may contribute to the dynamics of graviperception by influencing amyloplast movement.

  20. Life as a moving fluid: fate of cytoplasmic macromolecules in dynamic fungal syncytia

    PubMed Central

    Roper, Marcus; Lee, ChangHwan; Hickey, Patrick C.; Gladfelter, Amy S.

    2015-01-01

    In fungal syncytia dozens, or even millions of nuclei may coexist in a single connected cytoplasm. Recent discoveries have exposed some of the adaptations that enable fungi to marshall these nuclei to produce complex coordinated behaviors, including cell growth, nuclear division, secretion and communication. In addition to shedding light on the principles by which syncytia (including embryos and osteoplasts) are organized, fungal adaptations for dealing with internal genetic diversity and physically dynamic cytoplasm may provide mechanistic insights into how cells generally are carved into different functional compartments. In this review we focus on enumerating the physical constraints associated with maintaining macromolecular distributions within a fluctuating and often flowing cytoplasmic interior. PMID:26226449

  1. Geologic Map of the Mylitta Fluctus Quadrangle (V-61), Venus

    USGS Publications Warehouse

    Ivanov, Mikhail A.; Head, James W., III

    2006-01-01

    INTRODUCTION The Magellan Mission The Magellan spacecraft orbited Venus from August 10, 1990, until it plunged into the Venusian atmosphere on October 12, 1994. Magellan Mission objectives included: (1) improving knowledge of the geological processes, surface properties, and geologic history of Venus by analysis of surface radar characteristics, topography, and morphology, and (2) improving the knowledge of the geophysics of Venus by analysis of Venusian gravity. The Magellan spacecraft carried a 12.6-cm radar system to map the surface of Venus. The transmitter and receiver systems were used to collect three data sets: (1) synthetic aperture radar (SAR) images of the surface, (2) passive microwave thermal emission observations, and (3) measurements of the backscattered power at small angles of incidence, which were processed to yield altimetric data. Radar imaging, altimetric, and radiometric mapping of the Venusian surface was done in mission cycles 1, 2, and 3 from September 1990 until September 1992. Ninety-eight percent of the surface was mapped with radar resolution on the order of 120 meters. The SAR observations were projected to a 75-m nominal horizontal resolution, and these full-resolution data compose the image base used in geologic mapping. The primary polarization mode was horizontal-transmit, horizontal-receive (HH), but additional data for selected areas were collected for the vertical polarization sense. Incidence angles varied between about 20? and 45?. High resolution Doppler tracking of the spacecraft took place from September 1992 through October 1994 (mission cycles 4, 5, 6). Approximately 950 orbits of high-resolution gravity observations were obtained between September 1992 and May 1993 while Magellan was in an elliptical orbit with a periapsis near 175 km and an apoapsis near 8,000 km. An additional 1,500 orbits were obtained following orbit-circularization in mid-1993. These data exist as a 75? by 75? harmonic field.

  2. Molecular probes for identification of pathogenic viruses in mosquitoes.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Viral pathogens that cause disease in mosquitoes belong to three major groups: baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus); iridoviruses (MIVs) (Iridoviridae: Chloriridovirus); and cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus). Baculoviruses and iridoviruses are DNA vir...

  3. Current Status of Deltabaculoviruses, Cypoviruses and Chloriridoviruses Pathogenic for Mosquitoes.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are a variety of viral pathogens that cause disease in mosquitoes with most belonging to three major groups. The most common viruses of mosquitoes are the baculoviruses (DBVs) (Baculoviridae: Deltabaculovirus), cytoplasmic polyhedrosis viruses (CPVs) (Reoviridae: Cypovirus) and the iridovirus...

  4. Relation Between Basophilia and Fine Structure of Cytoplasm in the Fungus Allomyces macrogynus Em

    PubMed Central

    Blondel, Benigna; Turian, Gilbert

    1960-01-01

    In a fungus, Allomyces macrogynus Em., staining tests have revealed changes in the location of cytoplasmic basophilia following different phases of the developmental cycle. These variations in location were used to observe which fine structures correspond to basophile and non-basophile areas of the cytoplasm. Hyphae, gametangia, zygotes, and plants were fixed at various developmental stages in OsO4, pH 6.1, and embedded in vestopal. Sections were examined in the electron microscope. Comparison of basophile and non-basophile cytoplasms leads to the conclusion that cytoplasmic particles of 150 to 200 A in diameter are responsible for basophilia. The possibility of these particles being ribosomes is discussed and confirmed. The present paper also describes some observations on the fine structure of other cellular components of this fungus, such as nuclei, mitochondria, various granules, and flagella. PMID:13801597

  5. Hybridization using cytoplasmic male sterility and herbicide tolerance from nuclear genes

    SciTech Connect

    Beversdorf, W.D.; Erickson, L.R.; Grant, I.

    1987-04-14

    An improved process is described for producing a substantially homogeneous population of plants of a predetermined hybrid variety of a crop which is capable of undergoing both self-pollination and cross-pollination. This process comprises: growing in a first planting area a substantially random population of cytoplasmic male sterile plants which exhibit tolerance to at least one herbicide attributable solely to homozygous dominant nuclear genes, and male fertile plants which are capable of pollinating the cytoplasmic male sterile plants and which lack the herbicide tolerance because the presence of homozygous recessive nuclear genes for such trait. The cytoplasmic male sterile plants and the male fertile plants are pollinated with pollen derived from the male fertile plants. Seed is formed on the cytoplasmic male sterile plants and on the male fertile plants. Harvesting in bulk the seed is formed on the plants of the first planting area.

  6. Cytoplasm enhancement operator of peripheral blood smear images that are instable-stained and overexposed

    NASA Astrophysics Data System (ADS)

    Zheng, Xin; Wang, Guoyou; Liu, Jianguo

    2015-12-01

    Nucleus and cytoplasm are both essential for white blood cell recognition but the edges of cytoplasm are too blurry to be detected because of instable staining and overexposure. This paper aims at proposing a cytoplasm enhancement operator (CEO) to achieve accurate convergence of the active contour model. The CEO contains two parts. First, a nonlinear over-exposure enhancer map is yielded to correct over-exposure, which suppresses background noise while preserving details and improving contrast. Second, the over-exposed regions of cytoplasm in particular is further enhanced by a tri- modal histogram specification based on the scale-space filtering. The experimental results show that the proposed CEO and its corresponding GVF snake is superior to other unsupervised segmentation approaches.

  7. Novel nuclear-cytoplasmic interaction in wheat (Triticum aestivum) induces vigorous plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interspecific hybridization can be considered an accelerator of evolution, otherwise a slow process, solely dependent on mutation and recombination. Upon interspecific hybridization, several novel interactions between nuclear and cytoplasmic genomes emerge which provide additional sources of diversi...

  8. The microtubule nucleation activity of centrobin in both the centrosome and cytoplasm

    PubMed Central

    Shin, Wonjung; Yu, Nam-Kyung; Kaang, Bong-Kiun; Rhee, Kunsoo

    2015-01-01

    Centrobin resides in daughter centriole and play a critical role in centriole duplication. Nucleation and stabilization of microtubules are known biological activities of centrobin. Here, we report a specific localization of centrobin outside the centrosome. Centrobin was associated with the stable microtubules. In hippocampal cells, centrobin formed cytoplasmic dots in addition to the localization at both centrosomes with the mother and daughter centrioles. Such specific localization pattern suggests that cytoplasmic centrobin is not just a reserved pool for centrosomal localization but also has a specific role in the cytoplasm. In fact, centrobin enhanced microtubule formation outside as well as inside the centrosome. These results propose specific roles of the cytoplasmic centrobin for noncentrosomal microtubule formation in specific cell types and during the cell cycle. PMID:26083938

  9. Nonlinearity in cytoplasm viscosity can generate an essential symmetry breaking in cellular behaviors.

    PubMed

    Tachikawa, Masashi; Mochizuki, Atsushi

    2015-01-01

    The cytoplasms of ameboid cells are nonlinearly viscous. The cell controls this viscosity by modulating the amount, localization and interactions of bio-polymers. Here we investigated how the nonlinearity infers the cellular behaviors and whether nonlinearity-specific behaviors exist. We modeled the developed plasmodium of the slime mold Physarum polycephalum as a network of branching tubes and examined the linear and nonlinear viscous cytoplasm flows in the tubes. We found that the nonlinearity in the cytoplasm׳s viscosity induces a novel type of symmetry breaking in the protoplasmic flow. We also show that symmetry breaking can play an important role in adaptive behaviors, namely, connection of behavioral modes implemented on different time scales and transportation of molecular signals from the front to the rear of the cell during cellular locomotion. PMID:25261729

  10. Evolutionary genomics of nucleo-cytoplasmic large DNA viruses.

    PubMed

    Iyer, Lakshminarayan M; Balaji, S; Koonin, Eugene V; Aravind, L

    2006-04-01

    A previous comparative-genomic study of large nuclear and cytoplasmic DNA viruses (NCLDVs) of eukaryotes revealed the monophyletic origin of four viral families: poxviruses, asfarviruses, iridoviruses, and phycodnaviruses [Iyer, L.M., Aravind, L., Koonin, E.V., 2001. Common origin of four diverse families of large eukaryotic DNA viruses. J. Virol. 75 (23), 11720-11734]. Here we update this analysis by including the recently sequenced giant genome of the mimiviruses and several additional genomes of iridoviruses, phycodnaviruses, and poxviruses. The parsimonious reconstruction of the gene complement of the ancestral NCLDV shows that it was a complex virus with at least 41 genes that encoded the replication machinery, up to four RNA polymerase subunits, at least three transcription factors, capping and polyadenylation enzymes, the DNA packaging apparatus, and structural components of an icosahedral capsid and the viral membrane. The phylogeny of the NCLDVs is reconstructed by cladistic analysis of the viral gene complements, and it is shown that the two principal lineages of NCLDVs are comprised of poxviruses grouped with asfarviruses and iridoviruses grouped with phycodnaviruses-mimiviruses. The phycodna-mimivirus grouping was strongly supported by several derived shared characters, which seemed to rule out the previously suggested basal position of the mimivirus [Raoult, D., Audic, S., Robert, C., Abergel, C., Renesto, P., Ogata, H., La Scola, B., Suzan, M., Claverie, J.M. 2004. The 1.2-megabase genome sequence of Mimivirus. Science 306 (5700), 1344-1350]. These results indicate that the divergence of the major NCLDV families occurred at an early stage of evolution, prior to the divergence of the major eukaryotic lineages. It is shown that subsequent evolution of the NCLDV genomes involved lineage-specific expansion of paralogous gene families and acquisition of numerous genes via horizontal gene transfer from the eukaryotic hosts, other viruses, and bacteria